Methods and compositions for modulating ACE-2 activity

ABSTRACT

Binding polypeptides comprising specific amino acid sequences are disclosed that specifically bind ACE-2 protein or ACE-2-like polypeptides. The binding polypeptides can be used in methods of the invention for detecting, isolating, or purifying ACE-2 protein or ACE-2-like polypeptides in solutions or mixtures, or biological samples. The invention also relates to nucleic acid molecules encoding these ACE-2 binding polypeptides, vectors and host cells containing these nucleic acids, and methods for producing the same. The present invention also relates to methods and compositions for detecting, diagnosing, prognosing, preventing, treating or ameliorating a disease or disorder associated with aberrant ACE-2 or ACE-2 receptor expression or inappropriate function of ACE-2 or ACE-2 receptor, comprising use of ACE-2 binding polypeptides or fragments or variants thereof, that specifically bind to ACE-2.

[0001] This application claims benefit under 35 U.S.C. §119(e) of U.S.Patent Application No. 60/295,004, filed Jun. 4, 2001, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to polypeptides that regulateproduction of Angiotensin 1-9 via such mechanisms as, for example,inhibition of ACE-2. Such polypeptides have uses for example, in thedetection, isolation, and/or purification of ACE-2 and/or Angiotensin1-9. The invention also relates to nucleic acid molecules encoding theseACE-2 binding polypeptides, vectors and host cells containing thesenucleic acids, and methods for producing the same. The present inventionalso relates to methods and compositions for detecting, diagnosing, orprognosing a disease or disorder associated with aberrant ACE-2 orAngiotensin 1-9 expression or inappropriate function of ACE-2 orAngiotensin 1-9, comprising ACE-2 binding polypeptides or fragments orvariants thereof, that specifically regulate ACE-2 action. The presentinvention further relates to methods and compositions for preventing,treating or ameliorating a disease or disorder associated with aberrantACE-2 or Angiotensin 1-9 expression or inappropriate ACE-2 function orAngiotensin 1-9 function, comprising administering to an animal,preferably a human, an effective amount of one or more ACE-2 bindingpolypeptides or fragments or variants thereof, that specificallyregulate ACE-2.

BACKGROUND OF THE INVENTION

[0003] The renin-angiotensin system (RAS) plays an important role incirculatory homeostasis at both systemic and local levels. Angiotensinconverting enzyme (ACE), a 175 kD protein known to be widely distributedthroughout the cardiovascular system, has been long recognized as thekey enzyme in the generation of angiotensin II, a peptide that regulatesfluid balance, blood pressure and local blood flow in a number oftissues (Peach, M. J. Physiological Reviews 57:313-370 (1997)). As partof an ongoing strategy to establish genes associated with cardiovascularfunction via high throughput cDNA sequencing, we identified a member ofthe RAS family of enzymes, ACE-2, from human kidney. This enzyme wasalso identified in a variety of tissues by others (Donoghue et al.,Circulation Research 87:e1-e9 (2000), Tipinis et al., The Journal ofBiological Chemistry 275:33238-33243 (2000)). The unmodified ACE-2protein contains transmembrane and signal peptide domains, but unlikeACE, ACE-2 contains just one single extracellular Zn⁺² bindingmetalloprotease domain (Tipinis et al., The Journal of BiologicalChemistry 275:33238-33243 (2000)). ACE-2 mRNA has a more limitedexpression pattern than ACE (Donoghue et al., Circulation Research87:e1-e9 (2000)) and, remarkably, no detectable expression in lungs(unpublished data).

[0004] ACE-2 and related carboxypeptidases (Snyder et al., The Journalof Biological Chemistry 260:7857-7860 (1985); Kokkonen et al.,Circulation 95:1455-1463 (1997)) catalyze the removal of the C-terminalleucine from angiotensin I to form the nonapeptide angiotensin 1-9(A1-9) or des-Leu¹⁰-angiotensin I (Donoghue et al., Circulation Research87:e1-e9 (2000); Tipinis et al., The Journal of Biological Chemistry260:7857-7860 (2000); Snyder et al., The Journal of Biological Chemistry260: 7857-7860 (1985); Snyder et al., Biochemica et Biophysica Acta871:1-5 (1986)). Circulating A1-9 has been detected in vivo at levelstwice that of angiotensin II (Oparil et al., Circulation Research29:682-690 (1971); Johnson et al., Peptides 10:489-492 (1989)). In thecase of ACE-2, the above reaction is not blocked by captopril,lisinopril or enalaprilat (Donoghue et al., Circulation Research87:e1-e9 (2000); Tipinis et al., The Journal of Biological Chemistry275:33238-33243 (2000)). The unique expression profile of ACE-2,spectrum of its enzymatic activity and inhibitory effects of its productA1-9 on ACE have led to the speculation that ACE-2 functions to affectcirculatory homeostasis by promoting vasodilation (Donoghue et al.,Circulation Research 87:e1-e9 (2000); Snyder et al., The Journal ofBiological Chemistry 260:7857-7860 (1985)). However, A1-9 has been shownto be a weak vasoconstrictor in isolated rat aorta and have weak pressoractivity in anesthetized rats and dogs (Oparil et al., CirculationResearch 29:682-690 (1971)). Therefore, we hypothesized that one of thephysiologic roles of ACE-2 is to increase arterial pressure through theactions of its catabolic product, A1-9. As such, ACE-2 might be a validtarget for drug development in hypertension.

[0005] Accordingly, molecules that specifically bind ACE-2 would find avariety of uses in the study of ACE-2, angiotensin 1-9, and angiotensin,as well as ACE, and its known substrates: Angiotensin II, Angiotensin1-7, des-Asp, bradykinin, neurotensin, and Substance P. Further,molecules that specifically bind ACE-2 would also find a variety of usesin the manufacture and purification of ACE-2, ACE, angiotensin,angiotensin II, and/or Angiotensin 1-9 in commercial and medically purequantities, and in the development new therapeutic or diagnosticreagents. ACE-2 binding polypeptides may also find medical utility in,for example, the treatment of cardiovascular disorders (e.g.,hypertension, chronic heart failure, left ventricular failure, stroke,cerebral vasospasm after subarachnoid injury, atherosclerotic heartdisease, and retinal hemorrhage), renal disorders (e.g., renal veinthrombosis, kidney infarction, renal artery embolism, renal arterystenosis, and edema, hydronephritis), proliferative diseases ordisorders (e.g., vascular stenosis, myocardial hypertrophy, hypertrophyand/or hyperplasia of conduit and/or resistance vessels, myocytehypertrophy, and fibroblast proliferative diseases), inflammatorydiseases (e.g., SIRS (systemic Inflammatory Response Syndromes), sepsis,polytrauma, inflammatory bowl disease, acute and chronic pain,rheumatoid arthritis, and osteo arthritis), allergic disorders (e.g.,asthma, adult respiratory distress syndrome, wound healing, and scarformation), as well as several other disoders and/or diseases (e.g.,periodontal disease, dysmenorrhea, premature labor, brain edemafollowing focal injury, diffuse axonal injury, and reperfusion injury).

SUMMARY OF THE INVENTION

[0006] The present invention provides new polypeptides and families ofpolypeptides that specifically bind ACE-2 and/or ACE-2-likepolypeptides. In particular, the invention encompasses polypeptides thatspecifically bind to a polypeptide or polypeptide fragment of humanACE-2 (SEQ ID NOs:138 and/or 142).

[0007] In particular, the invention relates to ACE-2 bindingpolypeptides comprising, or alternatively consisting of, an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-136,preferably SEQ ID NOs:11-39, more preferably SEQ ID NOs:23-24 and 36-39,as referred to below, in Tables 1-2 and Example 1 below, and fragmentsand variants thereof.

[0008] In specific preferred embodiments, the ACE-2 binding polypeptidesof the invention bind ACE-2 and/or ACE-2-like polypeptides with highaffinity. In other embodiments, the ACE-2 binding polypeptides of theinvention reversibly bind ACE-2 and/or ACE-2-like polypeptides. In stillother embodiments, the ACE-2 binding polypeptides of the inventionirreversibly bind ACE-2 and/or ACE-2-like polypeptides.

[0009] The cysteine residues in certain polypeptides according to theinvention are believed to form a disulfide bond, which would cause thepolypeptide containing these cysteine residues to form a stable loopstructure under non-reducing conditions. Especially preferred ACE-2binding polypeptides of the invention are polypeptide molecules thatcomprise amino acid sequences that form stable loop structures or otherstable structures that bind ACE-2 or ACE-2-like polypeptides.

[0010] Preferred binding polypeptides specific for ACE-2 include twoseparated, invariant cyteine residues and are thus capable of forming acyclic strucure under non-reducing conditions via a disulfide bondformed between the cysteine side chains. Specific ACE-2 bindingpolypeptides according to the present invention include polypeptidescomprising amino acid sequences of the following general formulae I-X:

Z₁-X₁—A-X₂-X₃-C-X₄-X₅-F—Z₂ (SEQ ID NO:1);  I.

[0011] wherein,

[0012] Z₁ is a polypeptide of at least 2 amino acids;

[0013] X₁ is any amino acid except cysteine;

[0014] X₂ is L or M (preferably L);

[0015] X₃ is F or Y (preferably F);

[0016] X₄ is F, L, M, or V (preferably V);

[0017] X₅ is D or E;

[0018] Z₂ is a polypeptide of at least one amino acid or is absent; and

[0019] Z₁ contains at least one cysteine residue such that formation ofa disulfide bond with the invariant cysteine residue (C) forms a cyclicpeptide of six or ten amino acids.

Z₁-X₁-C-X₂-X₃-X₄-Z₂ (SEQ ID NO:2)  II.

[0020] wherein,

[0021] Z₁ is a polypeptide of at least six amino acids;

[0022] X₁ is F, M, W, or Y (preferably F or Y);

[0023] X₂ is F, I, L, M, or V (preferably F or L);

[0024] X₃ is D, E, or T (preferably D);

[0025] X₄ is F or M (preferably F);

[0026] Z₂ is a polypeptide of at least one amino acid or is absent; and

[0027] Z₁ contains at least one cysteine residue such that formation ofa disulfide bond with the invariant cysteine residue (C) forms a cyclicpeptide of eight or ten amino acids.

Z₁-X₁-X₂-X₃-C-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X ₁₃-C-Z₂ (SEQ IDNO:3)  III.

[0028] wherein,

[0029] Z₁ is a polypeptide of at least one amino acid or is absent;

[0030] X₁ is A, D, F, G, H, L, N, P, or S (preferably D);

[0031] X₂ is A, D, F, G, H, N, S, W, or Y (preferably D);

[0032] X₃ is D, E, H, L, M, or V (preferably D or E);

[0033] X₄ is D, E, G, N, R, Q, S, or V (preferably D or E);

[0034] X₅ is N, T, or W (preferably W);

[0035] X₆ is any amino acid except cysteine;

[0036] X₇ is any amino acid except cysteine;

[0037] X₈ is F, W, or Y (preferably F);

[0038] X₉ is any amino acid except cysteine;

[0039] X₁₀ is any amino acid except cysteine;

[0040] X₁₁ is any amino acid except cysteine;

[0041] X₁₂ is any amino acid except cysteine;

[0042] X₁₃ is any amino acid except cysteine; and

[0043] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-R-X₁-X₂-X₃-X₄-D-S-X₅-C-Z₂ (SEQ ID NO:4)  IV.

[0044] wherein,

[0045] Z₁ is a polypeptide of at least one amino acid or is absent;

[0046] X₁ is any amino acid except cysteine;

[0047] X₂ is any amino acid except cysteine;

[0048] X₃ is C, E, or S;

[0049] X₄ is K, L, or R (preferably R);

[0050] X₅ is A, R, or S (preferably R);

[0051] Z₂ is a polypeptide of at least one amino acid or is absent; and

[0052] wherein, if X₃ is cysteine (C), then Z₁ contains a C-terminalcysteine residue.

Z₁-C-X₁-X₂-X₃-D-C-X₄-Z₂ (SEQ ID NO:5)  V.

[0053] wherein,

[0054] Z₁ is a polypeptide of at least one amino acid or is absent;

[0055] X₁ is any amino acid except cysteine (preferably L, H, or M);

[0056] X₂ is N or T (preferably T);

[0057] X₃ is any amino acid except cystein (preferably D, M, N, or S);

[0058] X₄ is V or I (preferably V);

[0059] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-C-F-X₁-W-X₂-Z₂ (SEQ ID NO:6);  VI.

[0060] wherein,

[0061] Z₁ is a polypeptide of at least one amino acid or is absent;

[0062] X₁ is D or E;

[0063] X₂ is D or E;

[0064] Z₂ is a polypeptide of at least two amino acids and contains atleast one cysteine residue such that formation of a disulfide bond withthe invariant cysteine residue (C) forms a cyclic peptide of seven,eight or twelve amino acids.

Z₁-X₁-E-X₂-C-H-X₃-X₄-P-X₅-X₆-C-Z₂ (SEQ ID NO:7)  VII.

[0065] wherein,

[0066] Z₁ is a polypeptide of at least one amino acid or is absent;

[0067] X₁ is W or Y;

[0068] X₂ is any amino acid except cysteine;

[0069] X₃ is W or Y;

[0070] X₄ is any amino acid except cysteine;

[0071] X₅ is any amino acid except cysteine;

[0072] X₆ is any amino acid except cysteine; and

[0073] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-K-E-C-K-F-G-Y-X₁-X₂-C-L-X₃-W-Z₂ (SEQ ID NO:8)  VIII.

[0074] wherein,

[0075] Z₁ is a polypeptide of at least one amino acid or is absent;

[0076] X₁ is any amino acid except cysteine;

[0077] X₂ is any amino acid except cysteine;

[0078] X₃ is any amino acid except cysteine; and

[0079] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-X₁-X₂-C-X₃-X₄-W-X₅-X₆-P-C-Z₂ (SEQ ID NO:9)  IX.

[0080] wherein,

[0081] Z₁ is a polypeptide of at least one amino acid or is absent;

[0082] X₁ is D or H (preferably D);

[0083] X₂ is any amino acid except cysteine (preferably H, N, or W);

[0084] X₃ is G or is absent;

[0085] X₄ is T or N (preferably T);

[0086] X₅ is any amino acid except cysteine (preferably A, N, W, or Y);

[0087] X₆ is any amino acid except cysteine (preferably H, N, or Q); and

[0088] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-C-X₁-X₂-X₃-R-X₄-X₅-P-W-X₆-X₇-C-Z₂ (SEQ ID NO: 10)  X.

[0089] wherein,

[0090] Z₁ is a polypeptide of at least one amino acid or is absent;

[0091] X₁ is any amino acid except cysteine (preferably K, L, R, or S);

[0092] X₂ is A or P (preferably P);

[0093] X₃ is any amino acid except cysteine (preferably I, L, Q, or V);

[0094] X₄ is any amino acid except cysteine (preferably D, G, H, M, Q,or Y);

[0095] X₅ is any amino acid except cysteine (preferably D, F, K, S, orY);

[0096] X₆ is any amino acid except cysteine (preferably F, K, M, or W;most preferably W);

[0097] X₇ is any amino acid except cysteine (preferably A, F, K, R, orV); and

[0098] Z₂ is a polypeptide of at least one amino acid or is absent.

[0099] ACE-2 binding polypeptides of the present invention includepolypeptides comprising amino acid sequences selected from the groupconsisting of: SEQ ID NO: N R E C H A L F C M D F 40 S P T C R A L F C VD F 41 S E N C Q A L F C V D F 42 S P T C R A L F C V D F 43 L E M C E AL F C V E F 44 N P E C G A L F C M E F 45 D F G C N A M F C V E F 46 D QN C F A M Y C F E F 47 N D Y C T V F T G A L F C L D F 48 P N Q C G V DI W A L F C V D F 49 E G N C F L I G P W C F E F 50 E G N C F L I G P WC F E F 51 H I E C E E W G Y W C I E M 52 W E D C L W I G M M C V E F 53Y E D C I G H A L F C M T F 54 D D K C F G W A H F C F D F 55 G G Q C GT S Y L F C I D F 56 Y S G C A D M Y M F C I D F 57 G G Q C G T S Y L FC I D F 58 K F E C M P S S L F C V D F 59 D D Y C F N I S S Y S Y C F DF 60 L H D C F I Y A D Y E Y C F D F 61 N H H C L E F S S F E Y C F D F62 D N L C M S G G S F D Y C F D F 63 S D Y C V G N N A V T Y C F D F 64N L D C I Y L Q N H S Y C F D F 65 D D D C M M L P L T M F C F D F 66 YD N C L G L A N L N F C F D F 67 H L D C Y N L V D N M F C F D F 68 N WN C L G T N E L Q F C L D F 69 Y F A C T N N D S Y L F C L D F 70 Y N FC M L I G E R D Y C L D F 71 D D V C Y S L I M A D Y C L D F 72 Y F A CT N N D S Y L F C L D F 73 D D M C R W Y P F A S F Y M C L F - 74 D D HC E W A S Y W K W D L C L H D 75 D D V C E N A D F A W L G W C M H F 76D D D C G W I G F A N F H L C L H G 77 F D D C Q T S W F Q G F W L C I DD 78 F H D C S W G P W G P W E I C T R L 79 S N D C V W L Q F W G G D MC F L P 80 N A D C E W V N F N H V D L C M W N 81 G S D C E W V N F T MF Q M C I S N 82 A W D C E W N L F D S T F F C P G F 83 L Y E C E W K QF G P V E M C L N F 84 H S E C R W E W F G R T M I C M S F 85 S G E C NW Q Q F S G W E I C L R D 86 A Y L C D W I L F D S F E M C L A P 87 P FE C D W G P W T L E M L C G P P 88 R G H C R D S R C M M N A P G 89 R IG C R D S R C N W W A P G 90 R G F C R D S S C S F P 91 R G W C L D S RC K V F 92 F L F C R L A S R D S R C A S P 93 F N P C R L Q S R D S A CR F R 94 F F P C R A L E K D S R C S F F 95 H F S C R L P S L D S R C QL W 96 N D V C L N D D C V Y G 97 W P T C L T M D C V Y N 98 H Y N C H TN D C V V L 99 H L R C M T S D C I H F 100 W V L C F E W E D C D E K 101Y E Y C F E W E Q C W E K 102 G I F C F E W E T C Y Q A 103 P Q F C F EW E P C F - - 104 I G F C F E W E V C Y E G 105 S I Y C F D W E D C W DE 106 Y D W C F D W E Q C W D Q 107 V G F C F D W E P C D E L 108 M D FC F D W E E C W T N 109 N I F C F D W E P C H F G 110 F E I C F D W E VC H E Q 111 D Y L C F D W E A C W L S 112 Y A M C F D W D E C F L G 113W ?W C F E W E D W C L V E 114 Y Q F C F D W E T T C W L D 115 V Y F C FD W E Q D C D E M 116 F Q L C F D W E E E C E E S 117 W A V C F D W EN - C G D K 118 W Q F C F D W D L N C D L R 119 Y W F C F D W E E D A NG H C G G N 120 F L L C F D W D I D W E Y G C Q H H 121 Y E E C H W R PM A C S T H 122 W E V C H W A P M M C K H G 123 Y E F C H Y A P Q E C KH M 124 ?K E C K F G Y S ?C L A W 125 Q K E C K F G Y P H C L P W 126 EH N C T W W N P C W T T 127 M D H C T W Q P C V L K 128 W D H C N W A HP C S R K 129 S D W CGT W N N P C F H Q 130 R Y L C L P Q R D K P W K FC N W F 131 R L H C K P Q R Q S P W M K C Q H L 132 Y S H C S P L R Y YP W W K C T Y P 133 L H A C R P V R G D P W W A C T L G 134 G F T C S PI R M F P W F R C D L G 135 F S P C K A L R H S P W W V C P S G 136

[0100] ACE-2 binding polypeptide molecules of the invention may alsohave an amino terminal (N-terminal) capping or functional group, such asan acetyl group, which, for example, blocks the amino terminal aminogroup from undesirable reactions or is useful in linking the ACE-2binding polypeptide to another molecule, matrix, resin, or solidsupport. ACE-2 binding polypeptides of the invention may also have acarboxy terminal (C-terminal) capping or functional group, such as anamide group, which, for example, blocks the C-terminal carboxyl groupfrom undesirable reactions or provides a functional group useful inconjugating the binding polypeptide to other molecules, matrices,resins, or solid supports. Preferably, the N- and/or C-terminal cappinggroups are polypeptide linker molecules. An especially preferredC-terminal linker molecule that is useful for immobilizing an ACE-2binding polypeptide of the invention to a solid support orchromatographic matrix material comprises the amino acid sequencePro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 146).

[0101] The invention also encompasses ACE-2 binding polypeptides thathave been modified, for example, to increase or decrease the stabilityof the molecule, while retaining the ability to bind ACE-2 and/orACE-2-like polypeptides. An example of a modified ACE-2 bindingpolypeptide of the invention is a polypeptide in which one of twocysteine residues is substituted with a non-naturally occurring aminoacid that is capable of condensing with the remaining cysteine sidechain to form a stable thioether bridge, thereby generating a cyclicBLyS binding polypeptide. Such cyclic thioether molecules of syntheticpeptides may be routinely generated using techniques known in the art,e.g., as described in PCT publication WO 97/4625 1, incorporated hereinby reference.

[0102] In another embodiment, the invention provides ACE-2 bindingpolypeptides of the invention attached, coupled, linked or adhered to amatrix or resin or solid support. Techniques for attaching, linking oradhering polypeptides to matrices, resins and solid supports are wellknown in the art. Suitable matrices, resins or solid supports for thesematerials may be any composition known in the art to which an ACE-2binding polypeptide of the invention could be attached, coupled, linked,or adhered, including but not limited to, a chromatographic resin ormatrix, such as SEPHAROSE-4 FF agarose beads, the wall or floor of awell in a plastic microtiter dish, such as used in an enzyme-likedimmunosorbent assay (ELISA), or a silica based biochip. Materials usefulas solid supports on which to immobilize binding polypeptides of theinvention include, but are not limited to, polyacrylamide, agarose,silica, nitrocellulose, paper, plastic, nylon, metal, and combinationsthereof. An ACE-2 binding polypeptide of the invention may beimmobilized on a matrix, resin or solid support material by anon-covalent association or by covalent bonding, using techniques knownin the art. Preferably, an ACE-2 binding polypeptide of the invention isimmobilized on a chromatography material such as SEPHAROSE-4 FF agarose.In an even more preferred embodiment, an ACE-2 binding polypeptide ofthe invention is coupled to a chromatography material using a linkermolecule. A preferred linker molecule according to the present inventionis a polypeptide comprising the amino acid sequencePro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 146). Most preferably, theaffinity chromatography material of the invention comprises an ACE-2binding polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs:11-136, which is linked to achromatography material by a polypeptide linker molecule having theamino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 146).ACE-2 binding polypeptides of the invention attached, coupled, linked oradhered to a matrix or resin or other solid support are useful formethods of detecting, isolating and purifying ACE-2 and/or ACE-2-likepolypeptides as well as Angiotensin 1-9 and/or Angeiotensin 1-9-likepolypeptides, particularly for purification of ACE-2 and/or ACE-2-likepolypeptides as well as Angiotensin 1-9 and/or Angeiotensin 1-9-likepolypeptides by affinity chromatography.

[0103] In certain preferred embodiments, the ACE-2 binding polypeptidesof the present invention or phage displaying such binding polypeptides,irreversibly bind the ACE-2 protein in its native form.

[0104] In certain preferred embodiments, the ACE-2 binding polypeptidesof the present invention or phage displaying such binding polypeptides,reversibly bind the ACE-2 protein in its native form.

[0105] In a further embodiment, the present invention encompasses acomposition of matter comprising isolated nucleic acids, preferably DNA,encoding an ACE-2 binding polypeptide of the invention. In a specificembodiment, nucleic acid molecules of the invention encode an ACE-2binding polypeptide of the invention as provided in SEQ ID NOs: 1-136.In additional embodiments, nucleic acid molecules of the inventionencode a polypeptide variant or fragment of a polypeptide comprising anamino acid sequence of SEQ ID NOs: 1-136. In a further additionalembodiment, nucleic acid molecules of the invention encode an ACE-2binding polypeptide, the complementary strand of which nucleic acidhybridizes to a polynucleotide sequence encoding a polypeptide describedin Tables 1-2 and in Example 1 (SEQ ID NOs: 1-136), under stringentconditions, e.g., hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2× SSC/0.1% SDS at about 50-65° C., under highly stringentconditions, e.g., hybridization to filter-bound nucleic acid in 6× SSCat about 45° C. followed by one or more washes in 0.1× SSC/0.2% SDS atabout 68° C., or under other stringent hybridization conditions whichare known to those of skill in the art (see, for example, Ausubel, F. M.et al., eds. , 1989, Current Protocols in Molecular Biology, Vol. 1,Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New Yorkat pages 6.3.1-6.3.6 and 2.10.3).

[0106] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules encoding the ACE-2 bindingpolypeptides of the present invention (as well as fragments and variantsthereof), and to host cells containing the recombinant vectors, as wellas to methods of making such vectors and host cells. The inventionfurther provides for the use of such recombinant vectors in theproduction of ACE-2 binding polypeptides by recombinant techniques.

[0107] The ACE-2 binding polypeptides, nucleic acids, transformed hostcells, and genetically engineered viruses and phage of the invention(e.g., recombinant phage), have uses that include, but are not limitedto, the detection, isolation, and purification of ACE-2.

[0108] In another embodiment of the invention, recombinant bacteriophagedisplaying ACE-2 binding polypeptides on their surfaces are alsoprovided. Such phage may be routinely generated using techniques knownin the art and are useful, for example, as screening reagents andreagents for detecting ACE-2.

[0109] In another embodiment, an ACE-2 binding polypeptide of theinvention is used to detect or isolate ACE-2 or ACE-2-like polypeptidesin a solution. Such solutions include, but are not limited to, ACE-2 orACE-2-like polypeptides suspended or dissolved in water or a buffersolution as well as any fluid and/or cell obtained from an individual,biological fluid, body tissue, body cell, cell line, tissue culture, orother source which may contain ACE-2 or ACE-2-like polypeptides, suchas, cell culture medium, cell extracts, and tissue homogenates.Biological fluids include, but are not limited to, sera, plasma, lymph,blood, blood fractions, urine, synovial fluid, spinal fluid, saliva, andmucous.

[0110] In another embodiment, the present invention provides a methodfor detecting ACE-2 protein and/or ACE-2-like polypeptide in a solutioncomprising, contacting the solution with an ACE-2 binding polypeptide ofthe invention and detecting binding of ACE-2 or ACE-2-like polypeptideto the ACE-2 binding polypeptide. The ACE-2 binding polypeptide may beeither free or immobilized. Preferably, the ACE-2 binding polypeptide isa polypeptide immobilized on a solid surface or chromatographic materialor the well of a plastic microtiter assay dish.

[0111] Another embodiment of the present invention is a method forisolating ACE-2 protein and/or an ACE-2-like polypeptide from asolution, comprising:

[0112] (a) contacting the solution with an ACE-2 binding polypeptideunder conditions that permit binding of the ACE-2 and/or ACE-2-likepolypeptides to ACE-2 binding polypeptides, and

[0113] (b) recovering the ACE-2 and/or ACE-2-like polypeptides.

[0114] A further embodiment of the present invention is a method forisolating ACE-2 protein and/or an ACE-2-like polypeptide from asolution, comprising:

[0115] (a) contacting the solution with an ACE-2 binding polypeptideunder conditions that permit binding of the ACE-2 and/or ACE-2-likepolypeptides to ACE-2 binding polypeptides,

[0116] (b) separating the complex(es) formed by the ACE-2 bindingpolypeptide and ACE-2 and/or ACE-2-like polypeptides from othercomponents of the solution,

[0117] (c) dissociating the ACE-2 binding polypeptide from the ACE-2and/or ACE-2-like polypeptides, and

[0118] (d) recovering the dissociated ACE-2 and/or ACE-2-likepolypeptides.

[0119] In another embodiment, the invention provides kits containing abinding polypeptide of the invention for use in methods of detecting orisolating ACE-2 and/or ACE-2-like polypeptides.

[0120] The present invention also provides panels of ACE-2 bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants) whereinthe panel members correspond to one, two, three, four, five, ten,fifteen, twenty, or more different ACE-2 binding polypeptides of theinvention. The present invention further provides mixtures of ACE-2binding polypeptides, wherein the mixture corresponds to one, two,three, four, five, ten, fifteen, twenty, or more different ACE-2 bindingpolypeptides of the invention. The present invention also provides forcompositions comprising, or alternatively consisting of, one, two,three, four, five, ten, fifteen, twenty, or more ACE-2 bindingpolypeptides of the present invention (including molecules comprising,or alternatively consisting of, ACE-2 binding polypeptide fragments orvariants thereof). A composition of the invention may comprise, oralternatively consist of, one, two, three, four, five, ten, fifteen,twenty, or more amino acid sequences of one or more ACE-2 bindingpolypeptides or fragments or variants thereof. Alternatively, acomposition of the invention may comprise, or alternatively consist of,nucleic acid molecules encoding one or more ACE-2 binding polypeptidesof the invention.

[0121] The present invention further provides for fusion proteinscomprising an ACE-2 binding polypeptide (including molecules comprising,or alternatively consisting of, ACE-2 binding polypeptide fragments orvariants thereof) of the invention, and a heterologous polypeptide.Nucleic acid molecules encoding these fusion proteins are alsoencompassed by the invention. A composition of the present invention maycomprise, or alternatively consist of, one, two, three, four, five, ten,fifteen, twenty or more fusion proteins of the invention. Alternatively,a composition of the invention may comprise, or alternatively consistof, nucleic acid molecules encoding one, two, three, four, five, ten,fifteen, twenty or more fusion proteins of the invention.

[0122] The present invention also encompasses methods and compositionsfor detecting, diagnosing, prognosing, and/or monitoring diseases ordisorders associated with aberrant ACE-2 or ACE expression orinappropriate ACE-2 or ACE receptor function in an animal, preferably amammal, and most preferably a human, comprising, or alternativelyconsisting of, use of ACE-2 binding polypeptides (including moleculeswhich comprise, or alternatively consist of, ACE-2 binding polypeptidefragments or variants thereof) that specifically bind ACE-2. Diseasesand disorders which can be detected, diagnosed, prognosed and/ormonitored with the ACE-2 binding polypeptides of the invention include,but are not limited to, cardiovascular disorders (e.g., hypertension,chronic heart failure, left ventricular failure, stroke, cerebralvasospasm after subarachnoid injury, atherosclerotic heart disease, andretinal hemorrhage), renal disorders (e.g., renal vein thrombosis,kidney infarction, renal artery embolism, renal artery stenosis, andedema, hydronephritis), proliferative diseases or disorders (e.g.,vascular stenosis, myocardial hypertrophy, hypertrophy and/orhyperplasia of conduit and/or resistance vessels, myocyte hypertrophy,and fibroblast proliferative diseases), inflammatory diseases (e.g.,SIRS (systemic Inflammatory Response Syndromes), sepsis, polytrauma,inflammatory bowl disease, acute and chronic pain, rheumatoid arthritis,and osteo arthritis), allergic disorders (e.g., asthma, adultrespiratory distress syndrome, wound healing, and scar formation), aswell as several other disoders and/or diseases (e.g., periodontaldisease, dysmenorrhea, premature labor, brain edema following focalinjury, diffuse axonal injury, and reperfusion injury).

[0123] In specific embodiments, the present invention encompassesmethods and compositions for detecting, diagnosing, prognosing and/ormonitoring diseases or disorders for preventing, treating and/orameliorating diseases or disorders associated with hypertension (e.g.,accelerated hypertension, renal failure, vascular accidents, myocaridalinfarction, and stroke).

[0124] In other specific embodiments, the present invention encompassesmethods and compositions for detecting, diagnosing, prognosing and/ormonitoring diseases or disorders associated with hypotension (e.g.,shock, intracranial hypotension, and syncope).

[0125] The present invention further encompasses methods andcompositions for preventing, treating and/or ameliorating diseases ordisorders associated with aberrant ACE-2 or ACE expression orinappropriate ACE-2 or ACE function in an animal, preferably a mammal,and most preferably a human, comprising, or alternatively consisting of,administering to an animal in which such treatment, prevention oramelioration is desired one or more ACE-2 binding polypeptides(including molecules which comprise, or alternatively consist of, ACE-2binding polypeptide fragments or variants thereof) in an amounteffective to treat, prevent or ameliorate the disease or disorder.Diseases and disorders which can be prevented, treated, and/orameliorated with the ACE-2 binding polypeptides of the inventioninclude, but are not limited to, cardiovascular disorders (e.g.,hypertension, chronic heart failure, left ventricular failure, stroke,cerebral vasospasm after subarachnoid injury, atherosclerotic heartdisease, and retinal hemorrhage), renal disorders (e.g., renal veinthrombosis, kidney infarction, renal artery embolism, renal arterystenosis, and edema, hydronephritis), proliferative diseases ordisorders (e.g., vascular stenosis, myocardial hypertrophy, hypertrophyand/or hyperplasia of conduit and/or resistance vessels, myocytehypertrophy, and fibroblast proliferative diseases), inflammatorydiseases (e.g., SIRS (systemic Inflammatory Response Syndromes), sepsis,polytrauma, inflammatory bowl disease, acute and chronic pain,rheumatoid arthritis, and osteo arthritis), allergic disorders (e.g.,asthma, adult respiratory distress syndrome, wound healing, and scarformation), as well as several other disoders and/or diseases (e.g.,periodontal disease, dysmenorrhea, premature labor, brain edemafollowing focal injury, diffuse axonal injury, and reperfusion injury).

[0126] In specific embodiments, the present invention encompassesmethods and compositions (e.g., ACE-2 binding polypeptides thatantagonize ACE-2 activity) for preventing, treating and/or amelioratingdiseases or disorders associated with hypertension (e.g., acceleratedhypertension, renal failure, vascular accidents, myocaridal infarction,and stroke).

[0127] In a specific embodiment, this invention also provides a methodfor preventing, ameliorating, or treating diseases and/or disordersassociated with hypotension (e.g., shock, syncope, and intracranialhypotension) comprising, or alternatively consisting of, administeringto an animal in which such inhibition or reduction is desired, aneffective amount of angiotensin II, or an angiotensin II-like compound,and an effective amount of angiotensin 1-9, or an angiotensin 1-9-likecompound.

[0128] In specific embodiments, the present invention encompassesmethods and compositions (e.g., ACE-2 binding polypeptides thatantagonize ACE-2 or ACE activity) for preventing, treating and/orameliorating other diseases or disorders associated withvasoconstriction, comprising, or alternatively consisting of,administering to an animal in which such treatment, prevention, and/oramelioration is desired, an ACE-2 binding polypeptide in an amounteffective to treat, prevent and/or ameliorate the disease or disorder.

[0129] The present invention further encompasses methods andcompositions for inhibiting or reducing stenosis, including aorticstenosis, buttonhole stenosis, coronary ostial stenosis, double aorticstenosis, fish-mouth mitral stenosis, bronchial stenosis, hypertrophicpyloric stenosis, pyloric stenosis, infundibular stenosis, idiopathichypertrophic subaortic stenosis, idiopathic subglottic stenosis,pulmonary stenosis, muscular subaortic stenosis, laryngeal stenosis,mitral stenosis, supravalvar and subvalvar stenosis, subvalvular andsupravalvular stenosis, and tricuspid stenosis, comprising, oralternatively consisiting of, contacting an effective amount of ACE-2binding polypeptide, wherein the effective amount of ACE-2 bindingpolypeptide inhibits or reduces ACE-2 mediated enzymatic action.

[0130] The present invention further encompasses methods andcompositions for inhibiting or reducing pain, comprising, oralternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, an ACE-2 binding polypeptide in anamount effective to inhibit or reduce ACE-2 enzymatic activity.

[0131] The present invention further encompasses methods andcompositions for inhibiting or reducing inflammatory reactions invarious tissues comprising, or alternatively consisting of, contactingan effective amount of ACE-2 binding polypeptide, wherein the effectiveamount of ACE-2 binding polypeptide inhibits or reduces ACE-2 enzymaticactivity.

[0132] In a specific embodiment, the present invention encompassesmethods and compositions for inhibiting or reducing inflammatoryreactions in smooth muscle tissues comprising, or alternativelyconsisting of, contacting an effective amount of ACE-2 bindingpolypeptide, wherein the effective amount of ACE-2 binding polypeptideinhibits or reduces ACE-2 enzymatic activity.

[0133] The present invention further encompasses methods andcompositions for inhibiting or reducing abnormal histamine releasecomprising, or alternatively consisting of, administering to an animalin which such inhibition or reduction is desired, an ACE-2 bindingpolypeptide in an amount effective to inhibit or reduce ACE-2 enzymaticactivity.

[0134] In another embodiment, the present invention encompasses methodsand compositions for inhibiting or reducing vasoconstriction and/orother diseases or disorders associated with vasoconstriction comprising,or alternatively consisting of, administering to an animal in which suchinhibition or reduction is desired, an ACE-2 binding polypeptide in anamount effective to inhibit or reduce ACE-2 enzymatic activity.

[0135] In yet another embodiment, this invention also provides a methodfor reducing or inhibiting diseases and/or disorders associated withaberrant action of ACE-2 comprising, or alternatively consisting of,administering to an animal in which such inhibition or reduction isdesired, an ACE-2 binding polypeptide and an effective amount of an ACEinhibiting compound.

[0136] Additionally, this invention also provides a method for reducingor inhibiting diseases and/or disorders associated with aberrant actionof ACE comprising, or alternatively consisting of, administering to ananimal in which such inhibition or reduction is desired, an ACE-2binding polypeptide and an effective amount of an ACE inhibitingcompound.

BRIEF DESCRIPTION OF THE DRAWINGS

[0137]FIG. 1. Synergistic effects of Angiotensin 1-9 (A1-9) onangiotensin II (AII)-induced vasoconstriction, representative experiment(experiment 9 from Table 3). Data are presented as the mean percentageof the maximal contractile response as defined by full KCldepolarization following treatment with angiotensin peptides for fourseparate rings used to determine average response at each concentration.Generated tension was plotted against peptide concentration and theresulting concentration-response curves were subjected to regressionanalysis to determine whether the effects of the combination of A1-9 andangiotensin II produced additivity or synergy. A1-9 had no effect onvascular tone in this experiment, hence the predicted effect of thecombination that would be expected if A1-9 additively potentiatedAII-mediated vasoconstriction is effectively that of AII alone (solidline). The synergistic or supraadditive potentiation of AII-mediatedvasoconstriction by A1-9 is shown (squares and dotted line) as aleftward shift.

[0138]FIG. 2. The effect of A1-9 on (A) mean arterial pressure ±SEM inthe awake and freely-ranging rat (n=6 rats). Average mean arterial andpulse pressure as well as heart rate data were derived from the last 5min of a 20 min continuous intravenous infusion period. * indicates asignificant difference compared to vehicle-treated rats followinganalysis of variance and Dunnett's post-hoc test. A1-9 or angiotensin II(AII) continuous infusion doses are in μg/kg/min.

[0139]FIG. 3. The effect of A1-9 on angiotensin II-mediated pressorresponses in normotensive awake male rats. Data are reported as averagemean arterial pressure ±SEM (n=6 rats).

[0140]FIG. 4. Arterial pressure responses to ACE-2 inhibitor or inactivepeptide in spontaneously hypertensive rats for 2.5 min following bolusinjection. Data are reported as mean MAP ±SEM. Since ACE-2 and inactivepeptides were tested in different cohorts of rats, separate vehicletreatment groups are plotted. The inactive peptide treated group wasalso treated with ACE-2 inhibitory peptide at the end of the experimentfor control. * indicates significant difference between ACE-2 inhibitorand vehicle treatment (ANOVA followed by Dunnett's). † indicatessignificant difference between ACE-2 inhibitor and control peptidetreated groups. The control peptide had no effect on MAP.

DEFINITIONS

[0141] In order that the invention may be clearly understood, thefollowing terms are defined:

[0142] The term “recombinant” is used to describe non-naturally alteredor manipulated nucleic acids, host cells transfected with exogenousnucleic acids, or polypeptide molecules that are expressednon-naturally, through manipulation of isolated nucleic acid (typically,DNA) and transformation or transfection of host cells. “Recombinant” isa term that specifically encompasses nucleic acid molecules that havebeen constructed in vitro using genetic engineering techniques, and useof the term “recombinant” as an adjective to describe a molecule,construct, vector, cell, polypeptide or polynucleotide specificallyexcludes naturally occurring such molecules, constructs, vectors, cells,polypeptides or polynucleotides.

[0143] The term “bacteriophage” is defined as a bacterial viruscontaining a nucleic acid core and a protective shell built up by theaggregation of a number of different protein molecules. The terms“bacteriophage” and “phage” are synonymous and are used hereininterchangeably.

[0144] The term “affinity ligand” is sometimes used herein and issynonymous with ACE-2 binding polypeptides of the invention.

[0145] The term “ACE-2 protein” as used herein encompasses both themembrane (e.g., SEQ ID NOs: 138 and 142) and soluble forms (e.g., SEQ IDNO: 140). ACE-2 protein may be monomeric, dimeric, or trimeric ormultivalent, preferably, ACE-2 proteins are homotrimeric.

[0146] The term “ACE-2-like polypeptide” as used herein encompassesnatural ACE-2 or full-length recombinant ACE-2 as well as fragments andvariants thereof, such as, a modified or truncated form of natural ACE-2or full-length recombinant ACE-2, which ACE-2 and ACE-2-like polypeptideretain an ACE-2 functional activity. ACE-2 or ACE-2 fragments that maybe specifically bound and/or inhibited by the compositions of theinvention include, but are not limited to, the ACE-2 polypeptide asshown in SEQ ID NO:138, the ACE-2 polypeptide as shown in SEQ ID NO:142,the ACE-2 extracellular domain (SEQ ID NO:140), and the ACE-2transmembrane domain (SEQ ID NO:139). ACE-2 and ACE-2-like polypeptidesretain at least one functional activity of the natural or full-lengthACE-2, including but not limited to the following activities: cleavingangiotensin to angiotensin 1-9 and regulating the cleavage and/orsynthesis of bradykinin, kinetensin, tachykinin, neurotensin, SubstanceP, and endothelin. In a preferred embodiment, the ACE-2 and ACE-2-likepolypeptides retain the ability to cleave angiotensin to angiotesin 1-9.Assays that can be used to determine the functional activities of ACE-2or ACE-2 like polypeptides can readily be determined by one skilled inthe art (e.g., see assays disclosed in Moore et al., 1999, supra)“ACE-2-like polypeptides” also include fusion polypeptides in which allor a portion of ACE-2 is fused or conjugated to another polypeptide.ACE-2-like polypeptides that are fusion polypeptides retain at least onefunctional activity of ACE-2, preferably the ability to cleaveangiotensin to angiotensin 1-9 and regulate the cleavage and/orsynthesis of bradykinin, kinetensin, tachykinin, neurotensin, SubstanceP, and endothelin. In a preferred embodiment, the ACE-2 and ACE-2-likepolypeptides that are fusion polypeptides retain the ability to cleaveangiotensin to angiotensin 1-9. ACE-2 fusion polypeptides may be made byrecombinant DNA techniques in which a gene or other polynucleotidecoding sequence for ACE-2 or a fragment thereof is ligated in-frame(recombined) with the coding sequence of another protein or polypeptide.The resulting recombinant DNA molecule is then inserted into any of avariety of plasmid or phage expression vectors, which enable expressionof the fusion protein molecule in an appropriate eukaryotic orprokaryotic host cell. ACE-2 fusion polypeptides may be generated bysynthetic or semi-synthetic procedures as well.

[0147] The terms “ACE-2 target” or “ACE-2 target protein” are sometimesused herein and encompass ACE-2 and/or ACE-2-like polypeptides. Thus,the ACE-2 binding polypeptides of the invention bind “ACE-2 targetproteins” and can be used to bind, detect, remove, and/or purify “ACE-2target proteins.”

[0148] The term “binding polypeptide” is used herein to refer to anypolypeptide capable of forming a binding complex with another molecule,polypeptide, peptidomimetic or transformant.

[0149] A “ACE-2 binding polypeptide” is a molecule of the invention thatcan bind an ACE-2 target protein. Non-limiting examples of ACE-2 bindingpolypeptides of the invention are the polypeptide molecules having anamino acid sequence described herein (see SEQ ID NOs: 1-136). The termACE-2 binding polypeptide also encompasses ACE-2 binding fragments andvariants (including derivatives) of polypeptides having the specificamino acid sequences described herein (SEQ ID NOs: 1-136). By “variant”of an amino acid sequence as described herein is meant a polypeptidethat binds ACE-2, but does not necessarily comprise an identical orsimilar amino acid sequence of an ACE-2 binding polypeptide specifiedherein. ACE-2 binding polypeptides of the invention which are variantsof an ACE-2 binding polypeptide specified herein satisfy at least one ofthe following: (a) a polypeptide comprising, or alternatively consistingof, an amino acid sequence that is at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, least 99%, or 100% identical to the amino acidsequence of an ACE-2 binding polypeptide sequence disclosed herein (SEQID NOs: 1-136), (b) a polypeptide encoded by a nucleotide sequence, thecomplementary sequence of which hybridizes under stringent conditions toa nucleotide sequence encoding an ACE-2 binding polypeptide disclosedherein (e.g., a nucleic acid sequence encoding the amino acid sequenceof SEQ ID NOs: 1-136), and/or a fragment of an ACE-2 binding polypeptidedisclosed herein, of at least 5 amino acid residues, at least 10 aminoacid residues, at least 15 amino acid residues, or at least 20 aminoacid residues. ACE-2 binding polypeptides of the invention alsoencompass polypeptide sequences that have been modified for variousapplications provided that such modifications do not eliminate theability to bind an ACE-2 target. Specific, non-limiting examples ofmodifications contemplated include C-terminal or N-terminal amino acidsubstitutions or peptide chain elongations for the purpose of linkingthe ACE-2 bindor to a chromatographic material or other solid support.Other substitutions contemplated herein include substitution of one orboth of a pair of cysteine residues that normally form disulfide links,for example with non-naturally occurring amino acid residues havingreactive side chains, for the purpose of forming a more stable bondbetween those amino acid positions than the former disulfide bond. Allsuch modified binding polypeptides are also considered ACE-2 bindingpolypeptides according to this invention so long as the modifiedpolypeptides retain the ability to bind ACE-2 and/or ACE-2-likepolypeptides, and therefore, may be used in one or more of the variousmethods described herein, such as, to detect, purify, or isolate ACE-2or ACE-2-like polypeptides in or from a solution. ACE-2 bindingpolypeptides of the invention also include variants of the specificACE-2 binding polypeptide sequences disclosed herein (e.g., SEQ ID NOs:1-136) which have an amino acid sequence corresponding to one of thesepolypeptide sequences, but in which the polypeptide sequence is alteredby substitutions, additions or deletions that provide for moleucles thatbind ACE-2. Thus, the ACE-2 binding polypeptides include polypeptidescontaining, as a primary amino acid sequence, all or part of theparticular ACE-2 binding polypeptide sequence including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence, resulting in a peptidewhich is functionally active. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in asilent alteration. Conservative substitutions for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs. For example, the nonpolar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar-neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. -The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such ACE-2 binding polypeptidescan be made either by chemical peptide synthesis or by recombinantproduction from a nucleic acid encoding the ACE-2 binding polypeptidewhich nucleic acid has been mutated. Any technique for mutagenesis knownin the art can be used, including but not limited to, chemicalmutagenesis, in vitro site-directed mutagenesis (Hutchinson et al., J.Biol. Chem., 253:6551 (1978)), use of TAB.RTM. linkers (Pharmacia), etc.

[0150] As used and understood herein, percent homology or percentidentity of two amino acid sequences or of two nucleic acid sequences isdetermined using the algorithm of Karlin and Atschul (Proc. Natl. Acad.Sci. USA, 87: 2264-2268 (1990)), modified as in Karlin and Altschul(Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993)). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al. (J.Mol. Biol., 215: 403-410 (1990)). BLAST nucleotide searches areperformed with the NBLAST program to obtain nucleotide sequenceshomologous to a nucleic acid molecule described herein. BLAST proteinsearches are performed with the XBLAST program to obtain amino acidsequences homologous to a reference polypeptide. To obtain gappedalignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (Nucleic Acids Res., 25: 3389-3402 (1997)).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) are used. See,http://www.ncbi.nlm.nih.gov. Alternatively, the percent identity of twoamino acid sequences or of two nucleic acid sequences can be determinedonce the sequences are aligned for optimal comparison purposes (e.g.,gaps can be introduced in the sequence of a first amino acid or nucleicacid sequence for optimal alignment with a second amino acid or nucleicacid sequence). The amino acid residues or nucleotides at correspondingamino acid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide at the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % identity=number ofidentical overlapping positions/total number of positions×100%). In oneembodiment, the two sequences are the same length.

[0151] The term “polypeptide”, as used herein, refers to a linear,branched, or cyclic (e.g., containing a loop structure) polymer of twoor more amino acid residues linked with a peptide bond. The term“polypeptide” is not restricted to any particular upper limit of aminoacid residues. Thus, the ACE-2 affinity ligands of the invention thatcomprise an amino acid sequence described herein are properly referredto as “ACE-2 binding polypeptides” because such binding polypeptidescontain at least two amino acid residues held together by a peptidebond, even though such molecules may also contain one or more additionalmoieties or groups that are not amino acids, such as N-terminal and/orC-terminal capping or functional groups, and that may or may not beinvolved in a peptide bond. The polypeptides of the invention may bemonovalent, divalent, trivalent, or multivalent and may comprise one ormore of the ACE-2 binding polypeptides having the amino acid sequence ofSEQ ID NOs: 1-136 and/or fragments or variants thereof. The term“peptide” is used herein to have the same meaning as “polypeptide.” Theterm “antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. As such, the term antibody encompasses not only wholeantibody molecules, but also antibody fragments as well as variants(including derivatives) of antibodies and antibody fragments. Examplesof molecules which are described by the term “antibody” in thisapplication include, but are not limited to: single chain Fvs (scFvs),Fab fragments, Fab′ fragments, F(ab′)₂, disulfide linked Fvs (sdFvs),Fvs, and fragments comprising or alternatively consisting of, either aVL or a VH domain. The term “single chain Fv” or “scFv” as used hereinrefers to a polypeptide comprising a VL domain of antibody linked to aVH domain of an antibody.

[0152] “Feed stream”: ACE-2 and ACE-2-like polypeptides that are boundby an ACE-2 binding polypeptide of this invention may be produced by anymethod known in the art, including, but not limited to, chemicalsynthesis; production in transformed host cells; secretion into culturemedium by naturally occurring cells or recombinantly transformedbacteria, yeasts, fungi, insect cells, plant cells, and mammalian cells;production in genetically engineered organisms (for example, transgenicmammals); and production in non-genetically engineered organisms. Thesolution, sample, or mixture that contains an ACE-2 or ACE-2-likepolypeptide as it is produced or is found present in a productionsolution will sometimes be referred to as the “feed stream”.

[0153] The term “binding” refers to the determination by standardtechniques that a binding polypeptide recognizes and binds to a giventarget. Such standard techniques include, but are not limited to,affinity chromatography, equilibrium dialysis, gel filtration, enzymelinked immunosorbent assay (ELISA), FACS analysis, and the monitoring ofspectroscopic changes that result from binding, e.g., using fluorescenceanisotropy, either by direct binding measurements or competition assayswith another binder.

[0154] The term “specificity” refers to a binding polypeptide of theinvention that has a higher binding affinity for one target overanother. Thus, the term “ACE-2 target protein specificity” refers to amolecule having a higher affinity for ACE-2 target protein as comparedwith another molecule that is not an ACE-2 target protein.

[0155] The term “epitopes” as used herein refers to portions of ACE-2having antigenic or immunogenic activity in an animal, preferably amammal. An epitope having immunogenic activity is a portion of ACE-2that elicits an antibody response in an animal. An eptiope havingantigenic activity is a portion of ACE-2 to which an antibody or ACE-2binding polypeptide specifically binds as determined by any method knownin the art, for example, by the immunoassays described herein. Antigenicepitopes need not necessarily be immunogenic.

[0156] The term “fragment” as used herein refers to a polypeptidecomprising an amino acid sequence of at least 5 amino acid residues, atleast 6 amino acid residues, at least 7 amino acid residues, at least 8amino acid residues, at least 9 amino acid residues, at least 10 aminoacid residues, at least 11 amino acid residues, at least 12 amino acidresidues, at least 13 amino acid residues, at least 14 amino acidresidues, at least 15 amino acid residues, at least 16 amino acidresidues, at least 17 amino acid residues, at least 18 amino acidresidues, at least 19 amino acid residues, at least 20 amino acidresidues, at least 21 amino acid residues, at least 22 amino acidresidues, at least 23 amino acid residues, at least 24 amino acidresidues, or at least 25 amino acid residues of the amino acid sequenceof ACE-2, or an ACE-2 binding polypeptide (including molecules thatcomprise, or alternatively consist of, ACE-2 binding polypeptidefragments or variants thereof).

[0157] The term “fusion protein” as used herein refers to a polypeptidethat comprises, or alternatively consists of, an amino acid sequence ofan ACE-2 binding polypeptide of the invention and an amino acid sequenceof a heterologous polypeptide (i.e., a polypeptide unrelated to theACE-2 binding polypeptide).

[0158] The term “host cell” as used herein refers to the particularsubject cell transfected with a nucleic acid molecule and the progeny orpotential progeny of such a cell. Progeny may not be identical to theparent cell transfected with the nucleic acid molecule due to mutationsor environmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

[0159] Other terms are defined as necessary in the text below.

DETAILED DESCRIPTION OF THE INVENTION

[0160] The present invention provides novel binding moieties for ACE-2.Such binding moieties make possible the efficient detection andisolation of ACE-2 or ACE-2-like polypeptides in tissues or in asolution or system that contains ACE-2 or ACE-2-like polypeptides. TheACE-2 binding polypeptides disclosed herein can also be used toimmobilize ACE-2 targets and provide a means of removing ACE-2 targetproteins from solutions or systems containing them. The preferredbinding moieties of the present invention bind ACE-2 with high affinity,i.e., acting at low concentrations.

[0161] The present invention also encompasses methods and compositionsfor detecting, diagnosing, prognosing, and/or monitoring diseases ordisorders associated with aberrant ACE-2 or Angiotensin 1-9 expressionor inappropriate ACE-2 or Angiotensin 1-9 function in an animal,preferably a mammal, and most preferably a human, comprising, oralternatively consisting of, use of ACE-2 binding polypeptides(including molecules which comprise, or alternatively consist of, ACE-2binding polypeptide fragments or variants thereof) that specificallybind ACE-2. Diseases and disorders which can be detected, diagnosed,prognosed and/or monitored with the ACE-2 binding polypeptides of theinvention include, but are not limited to, cardiovascular disorders(e.g., hypertension, chronic heart failure, left ventricular failure,stroke, cerebral vasospasm after subarachnoid injury, atheroscleroticheart disease, and retinal hemorrhage), renal disorders (e.g., renalvein thrombosis, kidney infarction, renal artery embolism, renal arterystenosis, and edema, hydronephritis), proliferative diseases ordisorders (e.g., vascular stenosis, myocardial hypertrophy, hypertrophyand/or hyperplasia of conduit and/or resistance vessels, myocytehypertrophy, and fibroblast proliferative diseases), inflammatorydiseases (e.g., SIRS (systemic Inflammatory Response Syndromes), sepsis,polytrauma, inflammatory bowl disease, acute and chronic pain,rheumatoid arthritis, and osteo arthritis), allergic disorders (e.g.,asthma, adult respiratory distress syndrome, wound healing, and scarformation), as well as several other disoders and/or diseases (e.g.,periodontal disease, dysmenorrhea, premature labor, brain edemafollowing focal injury, diffuse axonal injury, and reperfusion injury).

[0162] Preferably, the present invention also encompasses methods andcompositions for detecting, diagnosing, prognosing, and/or monitoringdiseases or disorders including, but not limited to, hypertension anddiseases and/or disorders associated with hypertension, such asaccelerated hypertension, episodic hypertension, paroxysmalhypertension, portal hypertension, primary hypertension, secondaryhypertensoin, systemic venous hypertension, borderline hypertension,adrenal hypertension, benign hypertension, idiopathic hypertension, palehypertension, postpartm hypertension, pregnancy-induced hypertension(gestational hypertension), essential hypertension, labile hypertension,pulmonary hypertension, renal and renovascular hypertension, andGoldblatt hypertension, left ventricular failure, atherosclerotic heartdisease, stroke, retinal hemorrhage or infarction (Keith-Wagener-Barkerchanges), renal failure, renovascular disease, exudates, papilledema,vascular accidents, myocardial infarction, dissecting aneurysm.

[0163] The present invention further encompasses methods andcompositions for preventing, treating and/or ameliorating diseases ordisorders, especially diseases and disorders of vasoconstriction, oralternatively consisting of, administering to an animal in which suchtreatment, prevention or amelioration is desired one or more ACE-2binding polypeptides (including molecules which comprise, oralternatively consist of, ACE-2 binding polypeptide fragments orvariants thereof) in an amount effective to treat, prevent or amelioratethe disease or disorder. The present invention further encompassesmethods and compositions for preventing, treating and/or amelioratingdiseases or disorders associated with aberrant ACE-2 or Angiotensin 1-9expression or inappropriate ACE-2 or Angiotensin 1-9 function in ananimal, preferably a mammal, and most preferably a human, comprising, oralternatively consisting of, administering to an animal in which suchtreatment, prevention or amelioration is desired one or more ACE-2binding polypeptides (including molecules which comprise, oralternatively consist of, ACE-2 binding polypeptide fragments orvariants thereof) in an amount effective to treat, prevent or amelioratethe disease or disorder.

[0164] Diseases and disorders which can be prevented, treated, and/orameliorated with the ACE-2 binding polypeptides of the inventioninclude, but are not limited to, cardiovascular disorders (e.g.,hypertension, chronic heart failure, left ventricular failure, stroke,cerebral vasospasm after subarachnoid injury, atherosclerotic heartdisease, and retinal hemorrhage), renal disorders (e.g., renal veinthrombosis, kidney infarction, renal artery embolism, renal arterystenosis, and edema, hydronephritis), proliferative diseases ordisorders (e.g., vascular stenosis, myocardial hypertrophy, hypertrophyand/or hyperplasia of conduit and/or resistance vessels, myocytehypertrophy, and fibroblast proliferative diseases), inflammatorydiseases (e.g., SIRS (systemic Inflammatory Response Syndromes), sepsis,polytrauma, inflammatory bowl disease, acute and chronic pain,rheumatoid arthritis, and osteo arthritis), allergic disorders (e.g.,asthma, adult respiratory distress syndrome, wound healing, and scarformation), as well as several other disoders and/or diseases (e.g.,periodontal disease, dysmenorrhea, premature labor, brain edemafollowing focal injury, diffuse axonal injury, and reperfusion injury).

[0165] Preferably, the present invention also encompasses methods andcompositions for preventing, treating and/or ameliorating diseases ordisorders including, but not limited to, hypertension and diseasesand/or disorders associated with hypertension, such as acceleratedhypertension, episodic hypertension, paroxysmal hypertension, portalhypertension, primary hypertension, secondary hypertensoin, systemicvenous hypertension, borderline hypertension, adrenal hypertension,benign hypertension, idiopathic hypertension, pale hypertension,postpartm hypertension, pregnancy-induced hypertension (gestationalhypertension), essential hypertension, labile hypertension, pulmonaryhypertension, renal and renovascular hypertension, and Goldblatthypertension, left ventricular failure, atherosclerotic heart disease,stroke, retinal hemorrhage or infarction (Keith-Wagener-Barker changes),renal failure, renovascular disease, exudates, papilledema, vascularaccidents, myocardial infarction, dissecting aneurysm.

[0166] ACE-2 Binding Polypeptides

[0167] The present invention provides new polypeptides and families ofpolypeptides that specifically bind to ACE-2 (Angiotensin convertingenzyme-2) and/or ACE-2-like polypeptides. In particular, the inventionencompasses polypeptides that specifically bind to a polypeptide orpolypeptide fragment of human ACE-2 (SEQ ID NO: AAA).

[0168] In specific preferred embodiments, the ACE-2 binding polypeptidesof the invention bind ACE-2 and/or ACE-2-like polypeptides with highaffinity. In other embodiments, the ACE-2 binding polypeptides of theinvention reversibly bind ACE-2 and/or ACE-2-like polypeptides. In stillother embodiments, the ACE-2 binding polypeptides of the inventionirreversibly bind ACE-2 and/or ACE-2-like polypeptides.

[0169] In preferred embodiments, the ACE-2 binding polypeptides of thepresent invention (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants thereof),specifically bind to ACE-2 and do not cross-react with any otherantigens.

[0170] The cysteine residues in polypeptides are believed to form adisulfide bond, which would cause the polypeptide containing thesecysteine residues to form a stable loop structure under non-reducingconditions. Especially preferred ACE-2 binding polypeptides of theinvention are polypeptide molecules, which comprise amino acid sequencesthat form stable loop structures or other stable structures that bindACE-2 or ACE-2-like polypeptides.

[0171] In specific embodiments, the invention relates to ACE-2 bindingpolypeptides comprising, or alternatively consisting of, an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-136,preferably SEQ ID NOs:11-39, and most preferably SEQ ID NOs: 23-24 and36-39, as referred to in Tables 1-2 and in Example 1 below. Tenconsensus sequences (SEQ ID NOs:1-10) have been determined based on thespecific ACE-2 binding polypeptides shown in paragraph 530 of Example 1.In specific embodiments, ACE-2 binding polypeptides of the inventioncomprise one or more of these sequences. Such preferred ACE-2 bindingpolypeptides include a polypeptide with the potential to form a loopstructure comprising, or alternatively consisting of, an amino acidsequence selected from A-J (SEQ ID NOs:1-10):

Z₁-X₁-A-X₂-X₃-C-X₄-X₅-F-Z₂ (SEQ ID NO:1);  A.

[0172] wherein,

[0173] Z₁ is a polypeptide of at least 2 amino acids;

[0174] X₁ is any amino acid except cysteine;

[0175] X₂ is L or M (preferably L);

[0176] X₃ is F or Y (preferably F);

[0177] X₄ is F, L, M, or V (preferably V);

[0178] X₅ is D or E;

[0179] Z₂ is a polypeptide of at least one amino acid or is absent; and

[0180] Z₁ contains at least one cysteine residue such that formation ofa disulfide bond with the invariant cysteine residue (C) forms a cyclicpeptide of six or ten amino acids.

Z₁-X₁-C-X₂-X₃-X₄-Z₂ (SEQ ID NO:2)  B.

[0181] wherein,

[0182] Z₁ is a polypeptide of at least six amino acids;

[0183] X₁ is F, M, W, or Y (preferably F or Y);

[0184] X₂ is F, I, L, M, or V (preferably F or L);

[0185] X₃ is D, E, or T (preferably D);

[0186] X₄ is F or M (preferably F);

[0187] Z₂ is a polypeptide of at least one amino acid or is absent; and

[0188] Z₁ contains at least one cysteine residue such that formation ofa disulfide bond with the invariant cysteine residue (C) forms a cyclicpeptide of eight or ten amino acids.

Z₁-X₁-X₂-X₃-C-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-C-Z₂ (SEQ ID NO:3)  C.

[0189] wherein,

[0190] Z₁ is a polypeptide of at least one amino acid or is absent;

[0191] X₁ is A, D, F, G, H, L, N, P, or S (preferably D);

[0192] X₂ is A, D, F, G, H, N, S, W, or Y (preferably D);

[0193] X₃ is D, E, H, L, M, or V (preferably D or E);

[0194] X₄ is D, E, G, N, R, Q, S, or V (preferably D or E);

[0195] X₅ is N, T, or W (preferably W);

[0196] X₆ is any amino acid except cysteine;

[0197] X₇ is any amino acid except cysteine;

[0198] X₈ is F, W, or Y (preferably F);

[0199] X₉ is any amino acid except cysteine;

[0200] X₁₀ is any amino acid except cysteine;

[0201] X₁₁ is any amino acid except cysteine;

[0202] X₁₂ is any amino acid except cysteine;

[0203] X₁₃ is any amino acid except cysteine; and

[0204] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-R-X₁-X₂-X₃-X₄-D-S-X₅-C-Z₂ (SEQ ID NO:4)  D.

[0205] wherein,

[0206] Z₁ is a polypeptide of at least one amino acid or is absent;

[0207] X₁ is any amino acid except cysteine;

[0208] X₂ is any amino acid except cysteine;

[0209] X₃ is C, E, or S;

[0210] X₄ is K, L, or R (preferably R);

[0211] X₅ is A, R, or S (preferably R);

[0212] Z₂ is a polypeptide of at least one amino acid or is absent; and

[0213] wherein, if X3 is cysteine (C), then Z₁ contains a C-terminalcysteine residue.

Z_(-C-X) ₁-X₂-X₃-D-C-X₄-Z₂ (SEQ ID NO:5)  E.

[0214] wherein,

[0215] Z₁ is a polypeptide of at least one amino acid or is absent;

[0216] X₁ is L, H, or M;

[0217] X₂ is N or T (preferably T);

[0218] X₃ is D, M, N, or S;

[0219] X₄ is V or I (preferably V);

[0220] Z₂ is a polypeptide of at least one amino acid or is absent.

Z_(-C-F-X) ₁-W-X₂-Z₂ (SEQ ID NO:6);  F.

[0221] wherein,

[0222] Z₁ is a polypeptide of at least one amino acid or is absent;

[0223] X₁ is D or E;

[0224] X₂ is D or E;

[0225] Z₂ is a polypeptide of at least two amino acids and contains atleast one cysteine residue such that formation of a disulfide bond withthe invariant cysteine residue (C) forms a cyclic peptide of seven,eight or twelve amino acids.

Z₁-X₁-E-X₂-C-H-X₃-X₄-P-X₅-X₆-C-Z₂ (SEQ ID NO:7)  G.

[0226] wherein,

[0227] Z₁ is a polypeptide of at least one amino acid or is absent;

[0228] X₁ is W or Y;

[0229] X₂ is any amino acid except cysteine;

[0230] X₃ is W or Y;

[0231] X₄ is any amino acid except cysteine;

[0232] X₅ is any amino acid except cysteine;

[0233] X₆ is any amino acid except cysteine; and

[0234] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-K-E-C-K-F-G-Y-X₁-X₂-C-L-X₃-W-Z₂ (SEQ ID NO:8)  H.

[0235] wherein,

[0236] Z₁ is a polypeptide of at least one amino acid or is absent;

[0237] X₁ is any amino acid except cysteine;

[0238] X₂ is any amino acid except cysteine;

[0239] X₃ is any amino acid except cysteine; and

[0240] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-X₁-X₂-C-X₃-X₄-W-X₅-X₆-P-C-Z₂ (SEQ ID NO: 9)  I.

[0241] wherein,

[0242] Z₁ is a polypeptide of at least one amino acid or is absent;

[0243] X₁ is D or H (preferably D);

[0244] X₂ is H, N, or W;

[0245] X₃ is G or is absent;

[0246] X₄ is T or N (preferably T);

[0247] X₅ is A, N, W, or Y;

[0248] X₆ is H, N, or Q; and

[0249] Z₂ is a polypeptide of at least one amino acid or is absent.

Z₁-C-X₁-X₂-X₃-R-X₄-X₅-P-W-X₆-X₇-C-Z₂ (SEQ ID NO:10)  J.

[0250] wherein,

[0251] Z₁ is a polypeptide of at least one amino acid or is absent;

[0252] X₁ is K, L, R, or S;

[0253] X₂ is A or P (preferably P);

[0254] X₃ is I, L, Q, or V;

[0255] X₄ is D, G, H, M, Q, or Y;

[0256] X₅ is D, F, K, S, or Y;

[0257] X₆ is F, K, M, or W (preferably W);

[0258] X₇ is A, F, K, R, or V; and

[0259] Z₂ is a polypeptide of at least one amino acid or is absent.

[0260] wherein said polypeptides bind ACE-2 and/or ACE-2-likepolypeptides.

[0261] ACE-2 binding polypeptide molecules of the invention may alsohave an amino terminal (N-terminal) capping or functional group, such asan acetyl group, which, for example, blocks the amino terminal aminogroup from undesirable reactions or is useful in linking the ACE-2binding polypeptide to another molecule, matrix, resin, or solidsupport. The nature of the solid support, process for attachment of theACE-2 binding polypeptide to the solid support, solvent, and conditionsof the affinity isolation or selection procedure are largelyconventional and well known to those skilled in the art. ACE-2 bindingpolypeptides of the invention may also have a carboxy terminal(C-terminal) capping or functional group, such as an amide group, which,for example, blocks the C-terminal carboxyl group from undesirablereactions or provides a functional group useful in conjugating thebinding polypeptide to other molecules, matrices, resins, or solidsupports. Preferably, the N- and/or C-terminal capping groups arepolypeptide linker molecules. An especially preferred C-terminal linkermolecule that is useful for immobilizing an ACE-2 binding polypeptide ofthe invention to a solid support or chromatographic matrix materialcomprises the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQID NO: 146).

[0262] The invention also encompasses, ACE-2 binding polypeptides thathave been modified, for example, to increase or decrease the stabilityof the molecule, while retaining the ability to bind ACE-2 and/orACE-2-like polypeptides. An example of a modified ACE-2 bindingpolypeptide of the invention is a polypeptide in which one of twocysteine residues is substituted with a non-naturally occurring aminoacid that is capable of condensing with the remaining cysteine sidechain to form a stable thioether bridge, thereby generating a cyclicACE-2 binding polypeptide. Such cyclic thioether molecules of syntheticpeptides may be routinely generated using techniques known in the artand described, e.g., in PCT publication WO 97/46251, incorporated hereinby reference.

[0263] In another embodiment, the invention provides ACE-2 bindingpolypeptides of the invention attached, coupled, linked or adhered to amatrix or resin or solid support. Techniques for attaching linking oradhering polypeptides to matrices, resins and solid supports are wellknown in the art. Suitable matrices, resins or solid supports for thesematerials may be any composition known in the art to which bindingpolypeptides are commonly attached, coupled, linked, or adhered,including but not limited to, a chromatographic resin or matrix, such asSEPHAROSE-4 FF agarose beads, the wall or floor of a well in a plasticmicrotiter dish, such as used in an enzyme-liked immunosorbent assay(ELISA), or a silica based biochip. Materials useful as solid supportson which to immobilize binding polypeptides of the invention include,but are not limited to, polyacrylamide, agarose, silica, nitrocellulose,paper, plastic, nylon, metal, and combinations thereof. An ACE-2 bindingpolypeptide of the invention may be immobilized on a matrix, resin orsolid support material by a non-covalent association or by covalentbonding, using techniques known in the art. Preferably, an ACE-2 bindingpolypeptide of the invention is immobilized on SEPHAROSE-4 FF agarosechromatographic material. More preferably, an ACE-2 binding polypeptideof the invention is coupled to a chromatography material such asSEPHAROSE-4FF (agarose). In an even more preferred embodiment, an ACE-2binding polypeptide of the invention is coupled to a chromatographymaterial using a linker molecule. A preferred linker molecule accordingto the present invention is a polypeptide comprising the amino acidsequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO: 146). Mostpreferably, the affinity chromatography material of the inventioncomprises an ACE-2 binding polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:23-24 and 36-39, whichis linked to a chromatography material by a polypeptide linker moleculehaving the amino acid sequence Pro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ IDNO: 146). ACE-2 binding polypeptides of the invention attached, coupled,linked or adhered to a matrix or resin or solid support are useful formethods of detecting, isolating and purifying ACE-2 and/or ACE-2 likepolypeptides, particularly for purification of ACE-2 and/or ACE-2-likepolypeptides by affinity chromatography.

[0264] In certain preferred embodiments, the ACE-2 binding polypeptidesof the present invention or phage displaying such inhibitorypolypeptides, irreversibly inhibit the ACE-2 protein in its native form.

[0265] In certain preferred embodiments, the ACE-2 binding polypeptidesof the present invention or phage displaying such inhibitingpolypeptides, reversibly inhibit the ACE-2 protein in its native form.

[0266] ACE-2 binding polypeptides of the invention inhibit ACE-2 targetprotein with high affinity. In specific embodiments, ACE-2 bindingpolypeptides of the invention bind ACE-2 target proteins with adissociation constant or K_(D) of less than or equal to 5×10⁻² M, 10⁻²M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, or 10⁻⁵ M. Morepreferably, ACE-2 binding polypeptides of the invention bind ACE-2target proteins with a dissociation constant or K_(D) less than or equalto 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, or 10⁻⁸ M. Even morepreferably, ACE-2 binding polypeptides of the invention bind ACE-2target proteins with a dissociation constant or K_(D) less than or equalto 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

[0267] In certain preferred embodiments, ACE-2 binding polypeptides ofthe invention reversibly bind ACE-2 and/or ACE-2-like polypeptides andrelease bound ACE-2 protein in an active form, preferably in the nativeform, under specific release conditions. In specific embodiments, ACE-2binding polypeptides of the invention bind ACE-2 target proteins withoff-rates or k_(off) less than or equal to 10⁻¹ s⁻¹, 5×10⁻¹ s⁻¹, 10⁻²s⁻¹, 5×10⁻² s⁻¹, 10⁻³ s⁻¹, or 5×10⁻³ s⁻¹. More preferably, ACE-2 bindingpolypeptides of the invention bind ACE-2 target proteins with off-ratesor k_(off) less than or equal to, 10⁻⁴ s⁻¹, 5×10⁻⁴ s⁻¹, 10⁻⁵ s⁻¹, 5×10⁻⁵s⁻¹, 10⁻⁶ s⁻¹, 5×10⁻⁶ s⁻¹, 10⁻⁷ s⁻¹, or 5×10⁻⁷ s⁻¹.

[0268] Binding experiments to determine K_(D) and off-rates can beperformed in a number of conditions including, but not limited to, [pH6.0, 0.01% Tween 20], [pH 6.0, 0.1% gelatin], [pH 5.0, 0.01% Tween 20],[pH 9.0, 0.1% Tween 20], [pH 6.0, 15% ethylene glycol, 0.01% Tween20],[pH 5.0, 15% ethylene glycol, 0.01% Tween 20], and [pH 9.0, 15% ethyleneglycol, 0.01% Tween 20] The buffers in which to make these solutions canreadily be determined by one of skill in the art, and depend largely onthe desired pH of the final solution. Low pH solutions (<pH 5.5) can bemade, for example, in citrate buffer, glycine-HCl buffer, or in succinicacid buffer. High pH solutions can be made, for example, in Tris-HCl,phosphate buffers, or sodium bicarbonate buffers. A number of conditionsmay be used to determine K_(D) and off-rates for the purpose ofdetermining, for example, optimal pH and/or salt concentrations.

[0269] In certain embodiments, ACE-2 binding polypeptides of theinvention reversibly bind ACE-2 and/or ACE-2-like polypeptides,preferably in the native form.

[0270] In preferred embodiments, ACE-2 binding polypeptides of theinvention reversibly bind only the native form of ACE-2.

[0271] In certain embodiments, ACE-2 binding polypeptides of theinvention irreversibly bind ACE-2 and/or ACE-2-like polypeptides,preferably in the native form.

[0272] In preferred embodiments, ACE-2 binding polypeptides of theinvention irreversibly bind only the native form of ACE-2.

[0273] In some screening or assay procedures, it is possible and moreconvenient to use recombinant bacteriophage that display a particularACE-2 binding polypeptide instead of using isolated ACE-2 bindingpolypeptide. Such procedures include phage-based ELISA protocols andimmobilization of phage displaying a binding polypeptide tochromatographic materials. Such screening assays and procedures areroutine in the art and may be readily adapted for procedures using therecombinant bacteriophage of the present invention.

[0274] The invention also encompasses ACE-2 binding polypeptides thatcompetitively inhibit the binding of an ACE-2 binding polypeptidedisclosed herein (e.g., a polypeptide having the amino acid sequence ofSEQ ID NOS:11-39) for binding to ACE-2. Competitive inhibition can bedetermined by any suitable method known in the art, for example, usingthe competitive binding assays described herein. In preferredembodiments, the polypeptide competitively inhibits the binding of anACE-2 binding polypeptide disclosed herein (e.g., a polypeptide havingthe amino acid sequence of SEQ ID NOS:11-39) to ACE-2 by at least 95%,at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, or at least 50%. In a more preferred embodiment, the ACE-2binding polypeptide competitively inhibits the binding of an ACE-2binding polypeptide disclosed herein (e.g., a polypeptide having theamino acid sequence of SEQ ID NOS:11-39) to the native form of ACE-2, byat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50%.

[0275] In a further embodiment, the present invention encompasses acomposition of matter comprising isolated nucleic acids, preferably DNA,encoding an ACE-2 binding polypeptide of the invention. In a specificembodiment, nucleic acid molecules of the invention encode an ACE-2binding polypeptide of the invention as provided in SEQ ID NOs: 1-136.In additional embodiments, nucleic acid molecules of the inventionencode a polypeptide variant or fragment of a polypeptide having anamino acid sequence of SEQ ID NOs: 1-136. In a further additionalembodiment, nucleic acid molecules of the invention encode an ACE-2binding polypeptide, the complementary strand of which nucleic acidhybridizes to a polynucletide sequence encoding a polypeptide describedin Tables 1-2 and in Example 1 (SEQ ID NOs: 1-136), under stringentconditions, e.g., hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2× SSC/0.1% SDS at about 50-65° C., under highly stringentconditions, e.g., hybridization to filter-bound nucleic acid in 6× SSCat about 45° C. followed by one or more washes in 0.1× SSC/0.2% SDS atabout 68° C., or under other stringent hybridization conditions whichare known to those of skill in the art (see, for example, Ausubel etal., eds. , 1989, Current Protocols in Molecular Biology, Vol. I, GreenPublishing Associates, Inc. and John Wiley & Sons, Inc., New York atpages 6.3.1-6.3.6 and 2.10.3).

[0276] The present invention also relates to recombinant vectors thatinclude the isolated nucleic acid molecules encoding the ACE-2 bindingpolypeptides of the present invention (as well as fragments and variantsthereof), and to host cells containing the recombinant vectors, as wellas to methods of making such vectors and host cells. The inventionfurther provides for the use of such recombinant vectors in theproduction of ACE-2 binding polypeptides by recombinant techniques.

[0277] The ACE-2 binding polypeptides, nucleic acids, transformed hostcells, and genetically engineered viruses and phage of the invention(e.g., recombinant phage), have uses that include, but are not limitedto, the detection, isolation, and purification of ACE-2.

[0278] In another embodiment of the invention, recombinant bacteriophagedisplaying ACE-2 binding polypeptides on their surfaces are alsoprovided. Such phage may be routinely generated using techniques knownin the art and are useful, for example, as screening reagents andreagents for detecting ACE-2.

[0279] The invention also encompasses ACE-2 binding polypeptides(including molecules comprising, or alternatively consisting of, ACE-2binding polypeptide fragments or variants thereof) that have one or moreof the same biological characteristics as one or more of the ACE-2binding, polypeptides described herein. By “biological characteristics”is meant, the in vitro or in vivo activities or properties of the ACE-2binding polypeptides, such as, for example, the ability to bind to ACE-2(e.g., the soluble form of ACE-2, the membrane-bound form of ACE-2, thesoluble form and membrane-bound form of ACE-2), and/or an antigenicand/or epitope region of ACE-2, the ability to substantially block ACE-2enzymatic action, preferably the ability to substantially block ACE-2enzymatic action on Angiotensin, the ability to regulate ACE-2 mediatedbiological activity (e.g., production of Angiotensin II). Optionally,the ACE-2 binding polypeptides of the invention will bind to the sameepitope as at least one of the ACE-2 binding polypeptides specificallyreferred to herein. Such epitope binding can be routinely determinedusing assays known in the art.

[0280] The present invention also provides for ACE-2 bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants thereof),that neutralize ACE-2 or a fragment thereof, said ACE-2 bindingpolypeptides comprising, or alternatively consisting of, a polypeptidehaving an amino acid sequence of any one of SEQ ID NOs:1-136, preferablyof SEQ ID NOs: 11-39, most preferably of SEQ ID NOs: 23-24 and 36-39, ora fragment or variant thereof. By an ACE-2 binding polypeptide that“neutralizes ACE-2 or a fragment thereof” is meant an ACE-2 bindingpolypeptide that inhibits (i.e., is effective to reduce or abolish) orabolishes the ability of ACE-2: cleaving Angiotensin I to Angiotensin1-9 and regulating the cleavage and/or synthesis of bradykinin,kinetensin, tachykinin, neurotensin, Substance P, and endothelin.Nucleic acid molecules encoding these ACE-2 binding polypeptides arealso encompassed by the invention.

[0281] The present invention also provides for ACE-2 bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants thereof),that inhibit or abolish ACE-2 mediated cleaving of Angiotensin asdetermined by any method known in the art such as, for example, theassays described in Example 9, infra, said ACE-2 binding polypeptidescomprising, or alternatively consisting of, a polypeptide having anamino acid sequence of any one of SEQ ID NOs:1-136, preferably of SEQ IDNOs: 11-39, most preferably of SEQ ID NOs: 23-24 and 36-39, or afragment or variant thereof. Nucleic acid molecules encoding these ACE-2binding polypeptides are also encompassed by the invention.

[0282] The present invention also provides: ACE-2 binding polypeptides(including molecules comprising, or alternatively consisting of, ACE-2binding polypeptide fragments or variants thereof), that specificallybind to the soluble form of ACE-2; ACE-2 binding polypeptides thatspecifically bind to the membrane-bound form of ACE-2; and ACE-2 bindingpolypeptides that specifically bind to both the soluble form andmembrane-bound form of ACE-2.

[0283] The present invention also provides for mixtures of ACE-2 bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants thereof)that specifically bind to ACE-2, wherein the mixture contains at leastone, two, three, four, five or more different ACE-2 binding polypeptidesof the invention. In particular, the invention provides for mixtures ofdifferent ACE-2 binding polypeptides that specifically bind to thesoluble form of ACE-2, the membrane-bound form of ACE-2, and/or both themembrane-bound form and soluble form of ACE-2. In specific embodiments,the invention provides mixtures of at least 2, preferably at least 4, atleast 6, at least 8, at least 10, at least 12, at least 15, at least 20,or at least 25 different ACE-2 binding polypeptides that specificallybind to ACE-2, wherein at least 1, at least 2, at least 4, at least 6,or at least 10, ACE-2 binding polypeptides of the mixture are ACE-2binding polypeptides of the invention. In a specific embodiment, eachantibody of the mixture is an ACE-2 binding polypeptide of theinvention.

[0284] The present invention also provides for panels of ACE-2 bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants thereof)that specifically bind to ACE-2, wherein the panel has at least one,two, three, four, five or more different ACE-2 binding polypeptides ofthe invention. In particular, the invention provides for panels ofdifferent ACE-2 binding polypeptides that specifically bind to thesoluble form of ACE-2, the membrane-bound form of ACE-2, and/or both themembrane-bound form and soluble form of ACE-2. In specific embodiments,the invention provides for panels of ACE-2 binding polypeptides thathave different affinities for ACE-2, different specificities for ACE-2,or different dissociation rates. The invention provides panels of atleast 10, preferably at least 25, at least 50, at least 75, or at least100 ACE-2 binding polypeptides. Panels of ACE-2 binding polypeptides canbe used, for example, in 96 well plates for assays such as ELISAs.

[0285] The present invention further provides for compositionscomprising, one or more ACE-2 binding polypeptides (including moleculescomprising, or alternatively consisting of ACE-2 binding polypeptidefragments or variants of the invention). In one embodiment, acomposition of the present invention comprises, one, two, three, four,five, or more ACE-2 binding polypeptides that comprise or alternativelyconsist of, a polypeptide having an amino acid sequence of any one ormore of the ACE-2 binding polypeptides contained in SEQ ID NOs:1-136 asdisclosed in Tables 1-2 and Example 1, or a variant thereof.

[0286] As discussed in more detail below, a composition of the inventionmay be used either alone or in combination with other compositions. TheACE-2 binding polypeptides (including molecules comprising, oralternatively consisting of ACE-2 binding polypeptide fragments orvariants of the present invention) may further be recombinantly fused toa heterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, ACE-2 bindingpolypeptides of the present invention may be recombinantly fused orconjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, polypeptidelinkers, drugs, radionuclides, or toxins. See, e.g., PCT publications WO92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP396,387.

[0287] ACE-2 binding polypeptides of the present invention (includingmolecules comprising, or alternatively consisting of ACE-2 bindingpolypeptide fragments or variants of the present invention) may be used,for example, but not limited to, to purify and detect ACE-2, and totarget the polypeptides of the present invention to cells expressingmembrane-bound ACE-2 or ACE-2 receptor, including both in vitro and invivo diagnostic and therapeutic methods. For example, the ACE-2 bindingpolypeptides have use in immunoassays for qualitatively andquantitatively measuring levels of ACE-2 in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor 1988) (incorporated by referenceherein in its entirety).

[0288] Production and Modification of ACE-2 Binding Polypeptides

[0289] ACE-2 binding polypeptides of the invention may be produced bychemical synthesis, semi-synthetic methods, and recombinant DNAmethodologies known in the art.

[0290] In certain embodiments, ACE-2 binding polypeptides of the presentinvention are produced by chemical or semi-synthetic methodologies knownin the art (see, Kelley et al. in Genetic Engineering Principles andMethods, Setlow, J. K., ed. (Plenum Press, NY., 1990), vol. 12, pp.1-19; Stewart et al., Solid-Phase Peptide Synthesis, W. H. Freeman Co.,San Francisco, 1989). One advantage of these methodologies is that theyallow for the incorporation of non-natural amino acid residues into thesequence of the ACE-2 binding polypeptide.

[0291] In preferred embodiments, ACE-2 binding polypeptides of theinvention are chemically synthesized (see, e.g., Merrifield, J. Am.Chem. Soc., 85: 2149 (1963); Houghten, Proc. Natl. Acad. Sci. USA, 82:5132 (1985)). For example, polypeptides can be synthesized by solidphase techniques, cleaved from the resin, and purified by preparativehigh performance liquid chromatography (see, e.g., Creighton, Proteins:Structures and Molecular Properties (W. H. Freeman and Co., N.Y., 1983),pp. 50-60). ACE-2 binding polypeptides can also be synthesized by use ofa peptide synthesizer. The composition of the synthetic polypeptides maybe confirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; see Creighton, Proteins: Structures and MolecularProperties (W. H. Freeman and Co., N.Y., 1983), pp. 34-49). Furthermore,if desired, ACE-2 binding polypeptides of the invention may containnon-classical amino acids or chemical amino acid analogs, which canroutinely be introduced during chemical synthesis as a substitution oraddition into the ACE-2 binding polypeptides of the invention.Non-classical amino acids include, but are not-limited to, the D-isomersof the common amino acids, 2,4-diaminobutyric acid,alpha-aminoisobutyric acid, 4-aminobutyric acid (4Abu), 2-aminobutyricacid (Abu), 6-aminohexanoic acid (epsilon-Ahx), 2-aminoisobutyric acid(Aib), 3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,beta-alanine (bAla), fluoro-amino acids, designer amino acids such asbeta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

[0292] Solid phase peptide synthesis begins at the carboxy (C) terminusof the putative polypeptide by coupling a protected amino acid to asuitable resin, which reacts with the carboxyl group of the C-terminalamino acid to form a bond that is readily cleaved later, for example, ahalomethyl resin such as chloromethyl resin, bromomethyl resin,hydroxymethyl resin, aminomethyl resin, benzhydrylamine resin, ort-alkyloxycarbonyl-hydrazide resin. After removal of the α-aminoprotecting group with, for example, trifluoroacetic acid (TFA) inmethylene chloride and neutralization with, for example TEA, the nextcycle in the synthesis is ready to proceed. The remaining α-amino and,if necessary, side-chain-protected amino acids are then coupledsequentially in the desired order by condensation to obtain anintermediate compound connected to the resin. Alternatively, some aminoacids may be coupled to one another forming an oligopeptide prior toaddition to the growing solid phase polypeptide chain.

[0293] The condensation between two amino acids, or an amino acid and apeptide, or a peptide and a peptide can be carried out according tocondensation methods known in the art, including but not limited to, theazide method, mixed acid anhydride method, DCC(dicyclohexylcarbodiimide) method, active ester method (p-nitrophenylester method, BOP [benzotriazole-1-yl-oxy-tris (dimethylamino)phosphonium hexafluorophosphate] method, N-hydroxysuccinic acid imidoester method), and Woodward reagent K method.

[0294] Common to chemical synthesis of peptides is the protection orcapping (blocking) of the reactive side chain groups of the variousamino acid residues with suitable protecting or capping groups at thatsite until the group is ultimately removed after the polypeptide chainhas been completely assembled. Also common is the protection or cappingof the α-amino group on an amino acid or a fragment while that entityreacts at the carboxyl group followed by the selective removal of theα-amino-protecting group to allow subsequent reaction to take place atthat location. Accordingly, during synthesis, intermediate compounds areproduced which includes each of the amino acid residues located in thedesired sequence in the peptide chain with various of these residueshaving side-chain protecting or capping groups. These protecting orcapping groups on amino acid side chains are then removed substantiallyat the same time so as to produce the desired resultant productfollowing purification.

[0295] The typical protective, capping, or blocking groups for α- andε-amino side chain groups found in amino acids are exemplified bybenzyloxycarbonyl (Z), isonicotinyloxycarbonyl (iNOC),O-chlorobenzyloxycarbonyl [Z(NO₂)], p-methoxybenzyloxycarbonyl [Z(OMe)],t-butoxycarbonyl (Boc), t-amyioxycarbonyl (Aoc), isobornyloxycarbonyl,adamatyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc),9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonyiethoxycarbonyl (Msc),trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS),diphenylphosphinothioyl (Ppt), dimethylophosphinothioyl (Mpt), and thelike.

[0296] Protective, capping, or blocking groups for the carboxyl group ofamino acids include, for example, benzyl ester (OBzl), cyclohexyl ester(Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethylester (OPic), and the like. It is usually also desirable that side chaingroups of specific amino acids such as arginine, cysteine, and serine,are protected by a suitable protective group as occasion demands. Forexample, the guanidino group in arginine may be protected with nitro,p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl,p-methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Mds),1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group incysteine may be protected with p-methoxybenzyl, triphenylmethyl,acetylaminomethyl ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethy-benzyl(Tmb), etc., and the hydroxyl group in the serine can be protected withbenzyl, t-butyl, acetyl, tetrahydropyranyl, etc.

[0297] After the desired amino acid sequence has been completed, theintermediate polypeptide is removed from the resin support by treatmentwith a reagent, such as liquid HF and one or more thio-containingscavengers, which cleaves the peptide molecule from the resin and allthe remaining side-chain protecting groups. Following HF cleavage, theprotein sequence is washed with ether, transferred to a large volume ofdilute acetic acid, and stirred at pH adjusted to about 8.0 withammonium hydroxide. Upon pH adjustment, the polypeptide takes itsdesired conformational arrangement.

[0298] By way of example but not by way of limitation, polypeptides ofthe invention can be chemically synthesized and purified as follows:Peptides can be synthesized by employing theN-alpha-9-fluorenylmethyloxycarbonyl or Fmoc solid phase peptidesynthesis chemistry using a Rainin Symphony Multiplex PeptideSynthesizer. The standard cycle used for coupling of an amino acid tothe peptide-resin growing chain generally includes: (1) washing thepeptide-resin three times for 30 seconds with N,N-dimethylformamide(DMF); (2) removing the Fmoc protective group on the amino terminus bydeprotection to with 20% piperdine in DMF by two washes for 15 minuteseach, during which process mixing is effected by bubbling nitrogenthrough the reaction vessel for one second every 10 seconds to preventpeptide-resin settling; (3) washing the peptide-resin three times for 30seconds with DMF; (4) coupling the amino acid to the peptide resin byaddition of equal volumes of a 250 mM solution of the Fmoc derivative ofthe appropriate amino acid and an activator mix consisting or 400 mMN-methylmorpholine and 250 mM(2-(1H-benzotriazol-1-4))-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) in DMF; (5) allowing the solution to mix for 45 minutes; and (6)washing the peptide-resin three times for 30 seconds of DMF. This cyclecan be repeated as necessary with the appropriate amino acids insequence to produce the desired peptide. Exceptions to this cycleprogram are amino acid couplings predicted to be difficult by nature oftheir hydrophobicity or predicted inclusion within a helical formationduring synthesis. For these situations, the above cycle can be modifiedby repeating step 4 a second time immediately upon completion of thefirst 45 minute coupling step to “double couple” the amino acid ofinterest. Additionally, in the first coupling step in peptide synthesis,the resin can be allowed to swell for more efficient coupling byincreasing the time of mixing in the initial DMF washes to three 15minute washes rather than three 30 second washes.

[0299] After peptide synthesis, the peptide can be cleaved from theresin as follows: (1) washing the peptide-resin three times for 30seconds with DMF; (2) removing the Fmoc protective group on the aminoterminus by washing two times for 15 minutes it 20% piperdine in DMF;(3) washing the peptide-resin three times for 30 seconds with DMF; and(4) mixing a cleavage cocktail consisting of 95% trifluoroacetic acid(TFA), 2.4% water, 2.4% phenol, and 0.2% triisopropysilane with thepeptide-resin for two hours, then filtering the peptide in the cleavagecocktail away from the resin, and precipitating the peptide out ofsolution by addition of two volumes of ethyl ether. Specifically, toisolate the peptide, the ether-peptide solution can be allowed to sit at−20° C. for 20 minutes, then centrifuged at 6,000× G for 5 minutes topellet the peptide, and the peptide can be washed three times with ethylether to remove residual cleavage cocktail ingredients. The finalpeptide product can be purified by reversed phase high pressure liquidchromatography (RP-HPLC) with the primary solvent consisting of 0.1% TFAand the eluting buffer consisting of 80% acetonitrile and 0.1% TFA. Thepurified peptide can then be lyophilized to a powder.

[0300] In other specific embodiments, branched versions of the ACE-2binding polypeptides described herein are provided, e.g., bysubstituting one or more amino acids within the ACE-2 bindingpolypeptide sequence with an amino acid or amino acid analog with a freeside chain capable of forming a peptide bond with one or more aminoacids (and thus capable of forming a “branch”).

[0301] Branched peptides may be prepared by any method known in the artfor covalently linking any naturally occurring or synthetic amino acidto any naturally occurring or synthetic amino acid in a peptide chainwhich has a side chain group able to react with the amino or carboxylgroup on the amino acids so as to become covalently attached to thepeptide chain. In particular, amino acids with a free amino side chaingroup, such as, but not limited to, diaminobutyric acid, lysine,arginine, ornithine, diaminopropionic acid and citrulline, can beincorporated into a peptide so that an amino acid can form a branchtherewith, for example, by forming a peptide bond to the free amino sidegroup, from that residue. Alternatively, amino acids with a freecarboxyl side chain group, such as, but not limited to, glutamic acid,aspartic acid and homocitrulline, can be incorporated into the peptideso that an amino acid can form a branch therewith, for example, byforming a peptide bond to the free carboxyl side group, from thatresidue. The amino acid forming the branch can be linked to a side chaingroup of an amino acid in the peptide chain by any type of covalentbond, including, but not limited to, peptide bonds, ester bonds anddisulfide bonds. In a specific embodiment, amino acids, such as thosedescribed above, that are capable of forming a branch point, aresubstituted for ACE-2 binding polypeptide residues within a peptideincluding an ACE-2 binding polypeptide sequence.

[0302] Branched peptides can be prepared by any method known in the art.For example, but not by way of limitation, branched peptides can beprepared as follows: (1) the amino acid to be branched from the mainpeptide chain can be purchased as an N-alpha-tert-butyloxycarbonyl (Boc)protected amino acid pentafluorophenyl (Opfp) ester and the residuewithin the main chain to which this branched amino acid will be attachedcan be an N-Fmoc-alpha-gamma-diaminobutyric acid; (2) the coupling ofthe Boc protected amino acid to diaminobutyric acid can be achieved byadding 5 grams of each precursor to a flask containing 150 ml DMF, alongwith 2.25 ml pyridine and 50 mg dimethylaminopyridine and allowing thesolution to mix for 24 hours; (3) the peptide can then be extracted fromthe 150 ml coupling reaction by mixing the reaction with 400 mldichlormethane (DCM) and 200 ml 0.12N HCl in a I liter separatoryfunnel, and allowing the phases to separate, saving the bottom aqueouslayer and re-extracting the top layer two more times with 200 ml 0.12NHCl; (4) the solution containing the peptide can be dehydrated by adding2-5 grams magnesium sulfate, filtering out the magnesium sulfate, andevaporating the remaining solution to a volume of about 2-5 ml; (5) thedipeptide can then be precipitated by addition of ethyl acetate and then2 volumes of hexanes and then collected by filtration and washed twotimes with cold hexanes; and (6) the resulting filtrate can belyophilized to achieve a light powder form of the desired dipeptide.Branched peptides prepared by this method will have a substitution ofdiaminobutyric acid at the amino acid position which is branched.Branched peptides containing an amino acid or amino acid analogsubstitution other than diaminobutyric acid can be prepared analogouslyto the procedure described above, using the N-Fmoc coupled form of theamino acid or amino acid analog.

[0303] ACE-2 binding polypeptides a peptides of the invention may alsobe synthesized as multiple antigen peptides (MAPs). MAPs consist ofmultiple copies of a specific peptide attached to a non-immunogeniclysine core. By way of non-limiting example, MAPs may be synthesizedonto a lysine core matrix attached to a polyethylene glycol-polystyrene(PEG-PS) support. The peptide of choice is synthesized onto the lysineresidues using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. For example,PerSeptive Biosystems (Foster City, Calif.) offers MAP supports such asthe ([Fmoc-Lys(Aloc)] 4-[Lys] 2-Lys-Ala-PAl-PEG-PS) support which can beused to synthesize MAPs. Cleavage of MAPs from the resin is performedwith standard trifloroacetic acid (TFA)-based cocktails. Purification ofMAPs, except for desalting, is not necessary. In specific embodiments,ACE-2 binding polypetides of the invention being synthesized as MAPs maybe synthesized with additional C terminal “linker” residues. In morespecific embodiments, ACE-2 binding polypetides of the invention beingsynthesized as MAPs may be synthesized with an additional 6, 7, 8, 9,10, 11, 12, 13 or 14, C terminal residues. In even more specificembodiments, the additional residues are glycine and or serine residues.

[0304] ACE-2 binding polypeptides may be synthesized as MAPs in order tocreate multivalent ACE-2 binding polypeptides. By way of non-limitingexample, ACE-2 binding polypeptides synthesized as MAPs may be, forexample, labelled with a radiolabel using any method known in the art ordescribed herein, and used to label ACE-2 polypeptides. In a preferredembodiment, ACE-2 binding polypeptides synthesized as MAP peptidescomprising one or more DOTA molecules (see below) which are chelatingradiometal ions (e.g., ⁹⁰Y or ₁₁₁In) are used as a means ofradiolabelling ACE-2.

[0305] In another non-limiting example, ACE-2 binding polypeptides maybe synthesized as MAPs and used as an immunogen to create monoclonal orpolyclonal anti-ACE-2 binding polypeptide antibodies using any methodknown in the art or described herein.

[0306] In a preferred embodiment, divalent ACE-2 binding polypeptidesmay be synthesized by attaching two ACE-2 binding polypeptides to apolyethylene glycol (PEG) molecule. In one embodiment, the two ACE-2binding polypeptides attached to the PEG molecule are identical. Inanother embodiment, the two ACE-2 binding polypeptides attached to a PEGmolecule are different.

[0307] In a preferred embodiment, the ACE-2 binding polypeptide of theinvention is a cyclic peptide. Cyclization can be, for example, but notby way of limitation, via a disulfide bond between two cysteine residuesor via an amide linkage. For example, but not by way of limitation,disulfide bridge formation can be achieved by (1) dissolving thepurified peptide at a concentration of between 0.1-0.5 mg/ml in 0.01 Mammonium acetate, pH 7.5; (2) adding to the dissolved peptide 0.01 Mpotassium ferricyanide dropwise until the solution appears pale yellowin color and allowing this solution to mix for 24 hours; (3)concentrating the cyclized peptide to 5-10 ml of solution, repurifyingthe peptide by reverse phase-high pressure liquid chromatography(RP-HPLC) and finally lyophilizing the peptide. In a specificembodiment, in which the peptide does not contain two appropriatelysituated cysteine residues, cysteine residues can be introduced at theamino-terminus and/or carboxy-terminus and/or internally such that thepeptide to be cyclized contains two cysteine residues spaced such thatthe residues can form a disulfide bridge. Alternatively, a cyclicpeptide can be obtained by generating an amide linkage using, forexample but not limited to, the following protocol: An allyl protectedamino acid, such as aspartate, glutamate, asparagine or glutamine, canbe incorporated into the peptide as the first amino acid, and then theremaining amino acids are coupled on. The allyl protective group can beremoved by a two hour mixing of the peptide-resin with a solution oftetrakistriphenylphosphine palladium (0) in a solution of chloroformcontaining 5% acetic acid and 2.5% N-methylmorpholine. The peptide resincan be washed three times with 0.5% N,N-diisopropylethylamine (DIEA) and0.5% sodium diethyldithiocabamate in DMF. The amino terminal Fmoc groupon the peptide chain can be removed by two incubations for 15 minuteseach in 20% piperdine in DMF, and washed three times with DMF for 30seconds each. The activator mix, N-methylmorpholine and HBTU in DMF, canbe brought onto the column and allowed to couple the free amino terminalend to the carboxyl group generated by removal of the allyl group tocyclize the peptide. The peptide can be cleaved from the resin asdescribed in the general description of chemical peptide synthesis aboveand the peptide purified by reverse phase-high pressure liquidchromatography (RP-HPLC). In a specific embodiment, in which the peptideto be cyclized does not contain an allyl protected amino acid, an allylprotected amino acid can be introduced into the sequence of the peptide,at the amino-terminus, carboxy-terminus or internally, such that thepeptide can be cyclized.

[0308] In addition, according to certain embodiments, it is preferablethat the ACE-2 binding polypeptides of the invention are produced havingor retaining an amino terminal (N-terminal) and/or a carboxy terminal(C-terminal) capping group, which may protect the N-terminal orC-terminal amino acid from undesirable chemical reactions during use orwhich may permit further conjugations or manipulations of the bindingpolypeptide, for example, in conjugating the binding polypeptide to achromatographic support resin or matrix or to another peptide to tetherthe binding polypeptide to a resin or support. Such N-terminal andC-terminal groups may also be used to label or tag the bindingpolypeptide to detect bound complexes or to locate the bindingpolypeptide (whether bound or unbound to an ACE-2 target protein) forexample, at some point in a separation procedure. Accordingly, an ACE-2binding polypeptide of the invention synthesized in its final form foruse in a detection or separation procedure may contain an N-terminaland/or a C-terminal capping group. A particularly preferred N-terminalcapping group, which may be present or retained in binding polypeptidesof the invention, is an acetyl group (Ac). A particularly preferredC-terminal capping group, which may be present or retained in bindingpolypeptides of the invention, is an amide group. In a further preferredembodiment, the ACE-2 binding polypeptides of the invention have anacetyl group as an N-terminal capping group and an amide group as a Cterminal capping group.

[0309] The ACE-2 binding polypeptides of the invention may also beprepared commercially by companies providing polypeptide synthesis as aservice (e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; QualityControlled Biochemicals, Inc., Hopkinton, Mass.).

[0310] The nucleic acid sequence encoding an ACE-2 binding polypeptideof the invention can be produced and isolated using well-knowntechniques in the art. In one example, nucleic acids encoding the ACE-2binding polypeptides of the invention are chemically synthesized basedon knowledge of the amino acid sequence of the ACE-2 binding polypeptide(preferably the sequence is codon optimized to the host system in whichthe polypeptide will be expressed). In another example, nucleic acidsencoding an ACE-2 binding polypeptide are obtained by/screening anexpression library (e.g., a phage display library) to identify phageexpressing ACE-2 binding polypeptides, and isolating ACE-2 bindingpolypeptide encoding nucleic acid sequences from the identified librarymember (e.g., via polymerase chain reaction methodology using primersflanking the polypeptide encoding sequences).

[0311] The present invention also relates to vectors which includenucleic acid sequences encoding the ACE-2 binding polypeptides of theinvention, host cells which are genetically engineered with therecombinant vectors, or which are otherwise engineered to produce thepolypeptides of the invention, and the production of ACE-2 bindingpolypeptides, or fragments thereof, by recombinant, chemical orsynthetic techniques.

[0312] Thus, according to the present invention, ACE-2 bindingpolypeptidess can also be obtained by recombinant expression techniques.(See, e.g., Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d Ed., Glover, D. M. (ed.), (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989); DNA Cloning: A Practical Approach (MRLPress, Ltd., Oxford, U.K., 1985), Vols. I, II.

[0313] To produce a recombinant ACE-2 binding polypeptide, a nucleicacid sequence encoding the ACE-2 binding polypeptide is operativelylinked to a promoter such that the ACE-2 binding polypeptide is producedfrom said sequence. For example, a vector can be introduced into a cell,within which cell the vector or a portion thereof is expressed,producing the ACE-2 binding polypeptides. In a preferred embodiment, thenucleic acid is DNA if the source of RNA polymerase is DNA-directed RNApolymerase, but the nucleic acid may also be RNA if the source ofpolymerase is RNA-directed RNA polymerase or if reverse transcriptase ispresent in the cell or provided to produce DNA from the RNA. Such avector can remain episomal or, become chromosomally integrated, as longas it can be transcribed to produce the desired RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be bacteriophage, plasmid, viral, retroviral, or othersknown in the art, used for replication and expression in bacterial,fungal, plant, insect or mammalian cells. Retroviral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.Introduction of the vector construct into the host cell can be effectedby techniques known in the art which include, but are not limited to,calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0314] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, for example, stabilization or simplifiedpurification of expressed recombinant product.

[0315] In one embodiment, the nucleic acid encoding an ACE-2 bindingpolypeptide of the invention is operatively associated with anappropriate heterologous regulatory element (e.g., promoter orenhancer), such as, the phage lambda PL promoter, the E. coli lac, trp,phoA, and tac promoters, the SV40 early and late promoters, andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan.

[0316] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418, glutamine synthase or neomycin resistance foreukaryotic cell culture and tetracycline, kanamycin or ampicillinresistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, 293, NSO and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0317] The host cell can be a higher eukaryotic cell, such as amammalian cell (e.g., a human derived cell), or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. The host strain may be chosen which modulates theexpression of the inserted nucleic acid sequences encoding the ACE-2polypeptides of the invention, or modifies and processes the ACE-2binding polypeptide in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus expression of the genetically engineered polypeptide may becontrolled. Furthermore, different host cells have characteristics andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., phosphorylation, cleavage) ofproteins. Appropriate cell lines can be chosen to ensure the desiredmodifications and processing of the foreign protein expressed. Selectionof appropriate vectors and promoters for expression in a host cell is awell-known procedure and the requisite techniques for expression vectorconstruction, introduction of the vector into the host and expression inthe host are routine skills in the art.

[0318] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will preferablycomprise one or more phenotypic selectable markers and an origin ofreplication to ensure maintenance of the vector and to, if desirable,provide amplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice. As a representative, but nonlimiting example,useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well-known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1(Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. Among vectors preferred for use in bacteria are pHE4-5(ATCC Accession No. 209311) and variations thereof), pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Preferred expression vectors for use in yeast systemsinclude, but are not limited to, pYES2, pYD1, pTEF1/Zeo, pYES2/GS,pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K,pPIC9K, and PA0815 (all available from Invitrogen, Carlsbad, Calif.).Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL (availablefrom Pharmacia). Other suitable vectors will be readily apparent to theskilled artisan.

[0319] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

[0320] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well known to those skilled in the art.

[0321] In one embodiment, the yeast Pichia pastoris is used to expressan ACE-2 binding polypeptide in a eukaryotic system. Pichia pastoris isa methylotrophic yeast which can metabolize methanol as its sole carbonsource. A main step in the methanol metabolization pathway is theoxidation of methanol to formaldehyde using O₂. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O₂. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOXI gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis et al., Mol. Cell. Biol., 5:1111-21 (1985); Koutz et al., Yeast,5:167-77 (1989); Tschopp et al., Nucl. Acids Res., 15:3859-76 (1987).Thus, a heterologous coding sequence, such as, for example, apolynucleotide encoding a ACE-2 binding polypeptide of the presentinvention, under the transcriptional regulation of all or part of theAOX1 regulatory sequence is expressed at exceptionally high levels inPichia yeast grown in the presence of methanol.

[0322] In one example, the plasmid vector pPIC9K is used to express DNAencoding an ACE-2 binding polypeptide of the invention, as set forthherein, in a Pichea yeast system essentially as described in “PichiaProtocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg,eds. (The Humana Press, Totowa, N.J., 1998). This expression vectorallows expression and secretion of an ACE-2 binding polypeptide of theinvention by virtue of the strong AOX1 promoter linked to the Pichiapastoris alkaline phosphatase (PHO) secretory signal peptide (i.e.,leader) located upstream of a multiple cloning site.

[0323] Many other yeast vectors may be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S 1, pPIC3.5K, and PA0815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

[0324] In one embodiment, high-level expression of a heterologous codingsequence, such as, for example, a nucleic acid encoding an ACE-2 bindingpolypeptide of the invention, may be achieved by cloning theheterologous nucleic acid sequence of the invention into an expressionvector such as, for example, pGAPZ or pGAPZalpha, and growing the yeastculture in the absence of methanol.

[0325] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0326] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman (Cell, 23:175 (1981)), and other cell lines capable ofexpressing a compatible vector, for example, the C127, 3T3, CHO, 293,NSO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer, and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0327] Vectors which use glutamine synthase (GS) or DHFR as theselectable markers can be amplified in the presence of the drugsmethionine sulphoximine or methotrexate, respectively. An advantage ofglutamine synthase based vectors are the availabilty of cell lines(e.g., the murine myeloma cell line, NSO) which are glutamine synthasenegative. It is also possible to amplify vectors that utilize glutaminesynthase selection in glutamine synthase expressing cells (e.g., ChineseHamster Ovary (CHO) cells), however, by providing additional inhibitorto prevent the functioning of the endogenous gene. A glutamine synthaseexpression system and components thereof are detailed in PCTpublications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are hereby incorporated in their entireties byreference herein. Additionally, glutamine synthase expression vectorscan be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.).Expression and production of monoclonal antibodies using a GS expressionsystem in murine myeloma cells is described in Bebbington et al.,Bio/technology 10: 169(1992) and in Biblia and Robinson Biotechnol.Prog. 11:1 (1995) which are herein incorporated by reference.

[0328] The host cells described herein may be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.Alternatively, cell-free translation systems can also be employed toproduce the polypeptides of the invention using RNAs derived from theDNA constructs of the present invention.

[0329] The polypeptides of the invention may be expressed or synthesizedin a modified form, such as a fusion protein (comprising the polypeptidejoined via a peptide bond to a heterologous protein sequence (of adifferent protein)), and may include not only secretion signals, butalso additional heterologous functional regions. Such a fusion proteincan be made by ligating polynucleotides of the invention and the desirednucleic acid sequence encoding the desired amino acid sequence to eachother, by methods known in the art, in the proper reading frame, andexpressing the fusion protein product by methods known in the art.Alternatively, such a fusion protein can be made by protein synthetictechniques, e.g., by use of a peptide synthesizer. Thus, for instance, aregion of additional amino acids, particularly charged amino acids, maybe added to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. In preferred embodiments, the markeramino acid sequence is a hexa-histidine polypeptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA, 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the hemagglutinin “HA”tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., Cell, 37:767 (1984)) and the“flag” tag (DYKDDDDK, SEQ ID NO: 147) Stratagene, La Jolla, Calif.).

[0330] In one embodiment, polynucleotides encoding ACE-2 bindingpolypeptides of the invention may be fused to signal sequences whichwill direct the localization of a protein of the invention to particularcompartments of a prokaryotic or eukaryotic cell and/or direct thesecretion of a protein of the invention from a prokaryotic or eukaryoticcell. For example, in E. coli, one may wish to direct the expression ofthe protein to the periplasmic space. Examples of signal sequences orproteins (or fragments thereof) to which the polypeptides of theinvention may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pelB signal sequence, the maltose binding protein (MBP)signal sequence, MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit, and thesignal sequence of alkaline phosphatase. Several vectors arecommercially available for the construction of fusion proteins whichwill direct the localization of a protein, such as the pMAL series ofvectors (particularly the pMAL-p series) available from New EnglandBiolabs. In a specific embodiment, nucleic acids encoding ACE-2 bindingpolypeptides of the invention may be fused to the pelB pectate lyasesignal sequence to increase the efficiency to expression andpurification of such polypeptides in Gram-negative bacteria. See, U.S.Pat. Nos. 5,576,195 and 5,846,818, the contents of which are hereinincorporated by reference in their entireties.

[0331] Examples of signal peptides that may be fused to a polypeptide ofthe invention in order to direct its secretion in mammalian cellsinclude, but are not limited to, the MPIF-1 signal sequence (amino acids1-21 of GenBank Accession number AAB51134), the stanniocalcin signalsequence (MLQNSAVLLLLVISASA, SEQ ID NO:148), and a consensus signalsequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:149). A suitable signalsequence that may be used in conjunction with baculoviral expressionsystems is the gp67 signal sequence, (amino acids 1-19 of GenBankAccession Number AAA72759).

[0332] A preferred fusion protein comprises a heterologous region fromimmunoglobulin that is useful to stabilize and purify proteins. Forexample, EP-A-464 533 (Canadian counterpart 2,045,869) discloses fusionproteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A-232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when theFc portion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists. See, Bennettet al., J. Molecular Recognition, 8:52-58 (1995) and Johanson et al., J.Biol. Chem., 270:9459-9471 (1995).

[0333] In another preferred embodiment, ACE-2 binding polypetides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused with albumin (including but not limited torecombinant human serum albumin or fragments or variants thereof (see,e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622,and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporatedby reference in their entirety)). In a preferred embodiment,polypeptides and/or antibodies of the present invention (includingfragments or variants thereof) are fused with the mature form of humanserum albumin (i.e., amino acids 1-585 of human serum albumin as shownin FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated byreference in its entirety. In another preferred embodiment, polypeptidesand/or antibodies of the present invention (including fragments orvariants thereof) are fused with polypeptide fragments comprising, oralternatively consisting of, amino acid residues 1-z of human serumalbumin, where z is an integer from 369 to 419, as described in U.S.Pat. No. 5,766,883 herein incorporated by reference in its entirety.Polypeptides and/or antibodies of the present invention (includingfragments or variants thereof) may be fused to either the N- orC-terminal end of the heterologous protein (e.g., immunoglobulin Fcpolypeptide or human serum albumin polypeptide). Polynucleotidesencoding fusion proteins of the invention are also encompassed by theinvention.

[0334] The present invention encompasses ACE-2 binding polypeptides(including molecules comprising, or alternatively consisting of, ACE-2binding polypeptide fragments or variants thereof), recombinantly fusedor chemically conjugated (including both covalent and non-covalentconjugations) to a heterologous polypeptide (or portion thereof,preferably at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90 or at least 100amino acids of the heterologous polypeptide) to generate fusionproteins. The fusion does not necessarily need to be direct, but mayoccur through linker sequences. For example, ACE-2 binding polypeptidesof the invention may be used to target heterologous polypeptides toparticular cell types (e.g., smooth muscle cells, endothelial cells,cardiac cells, cardiovascular cells, testicular cells, and renal cells),either in vitro or in vivo, by fusing or conjugating the heterologouspolypeptides to ACE-2 binding polypeptides of the invention that arespecific for particular cell surface antigens (e.g., membrane-boundACE-2 on cells of cardiac myocytes and/or proximal tubules of thekidney) or which bind antigens (i.e., ACE-2 binding polypeptides) thatbind particular cell surface peptides (e.g., ACE-2 located on cardiacmyocytes, proximal convoluted tubules, endothelial cells, and/orepithelial cells of Bowman's capsule). ACE-2 binding polypeptides fusedor conjugated to heterologous polypeptides may also be used in in vitroimmunoassays and purification methods using methods known in the art.See e.g., Harbor et al., supra, and PCT publication WO 93/2 1232; EP 439095; Naramura et al., Immunol. Lett., 39:91-99 (1994); U.S. Pat. No.5,474,981; Gillies et al., Proc. Nat'l Acad. Sci. USA, 89:1428-1432(1992); Fell et al., J. Immunol., 146:2446-2452 (1991), which areincorporated by reference in their entireties.

[0335] The present invention further includes compositions comprising,or alternatively consisting of, heterologous polypeptides fused orconjugated to ACE-2 binding polypeptide fragment.

[0336] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofACE-2 binding polypeptides (including molecules comprising, oralternatively consisting of, ACE-2 binding polypeptide fragments orvariants thereof), such methods can be used to generate ACE-2 bindingpolypeptides with altered activity (e.g., ACE-2 binding polypeptideswith higher affinities and lower dissociation rates). See, generally,U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and5,837,458, and Patten et al., Curr. Opinion Biotechnol., 8:724-33(1997); Harayama, Trends Biotechnol., 16(2):76-82 (1998); Hansson, etal., J. Mol. Biol., 287:265-76 (1999); and Lorenzo and Blasco,Biotechniques, 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, polynucleotides encoding ACE-2 binding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more portions of apolynucleotide encoding an ACE-2 binding polypeptide which portionsspecifically bind to ACE-2 may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

[0337] Polypeptides of the present invention include products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

[0338] The invention encompasses ACE-2 binding polypeptides which aremodified during or after synthesis or translation, e.g., byglycosylation, acetylation, benzylation, biotinylation, phosphorylation,amidation, pegylation, formylation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule, hydroxylation, iodination, methylation, myristoylation,oxidation, pegylation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, ubiquitination,etc. (See, for instance, Creighton, Proteins: Structures and MolecularProperties, 2d Ed. (W. H. Freeman and Co., N.Y., 1992); PostranslationalCovalent Modification of Proteins, Johnson, ed. (Academic Press, NewYork, 1983), pp. 1-12; Seifter et al., Meth. Enzymol., 182:626-646(1990); Rattan et al., Ann. NY Acad. Sci., 663:48-62 (1992).) Inspecific embodiments, the peptides are acetylated at the N-terminusand/or amidated at the C-terminus.

[0339] In further embodiments, ACE-2 binding polypeptides of theinvention containing two or more residues that have the potential tointeract, such as for example, two cysteine residues in a polypeptide,may be treated under oxidizing conditions or other conditions thatpromote interaction of these residues (e.g., disulfide bridgeformation).

[0340] Further ACE-2 binding polypeptide modifications encompassed bythe invention include, for example, any of numerous chemicalmodifications carried out by known techniques, including but not limitedto specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formylation,oxidation, reduction, metabolic synthesis in the presence oftunicamycin, etc.

[0341] Additional post-translational/post-synthesis modificationsencompassed by the invention include, for example, e.g., N-linked orO-linked carbohydrate chains, processing of N-terminal or C-terminalends), attachment of chemical moieties to the amino acid backbone,chemical modifications of N-linked or O-linked carbohydrate chains, andaddition or deletion of an N-terminal methionine residue as a result ofprocaryotic host cell expression.

[0342] Also provided by the invention are chemically modifiedderivatives of ACE-2 binding polypetides of the invention which mayprovide additional advantages such as increased affinity, decreasedoff-rate, solubility, stability and in vivo or in vitro circulating timeof the polypeptide, or decreased immunogenicity (see, U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

[0343] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 1 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any, on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0344] As noted above, the polyethylene glycol may have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.,56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides, 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem., 10:638-646 (1999), thedisclosures of each of which are incorporated herein by reference.

[0345] The polyethylene glycol molecules (or other chemical moieties)should be attached to the ACE-2 binding poypeptide with consideration ofeffects on functional domains of the polypeptide. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol., 20:1028-1035 (1992) (reporting pegylationof GM-CSF using tresyl chloride). For example, polyethylene glycol maybe covalently bound through amino acid residues via a reactive group,such as, a free amino or carboxyl group. Reactive groups are those towhich an activated polyethylene glycol molecule may be bound. The aminoacid residues having a free amino group may include, for example, lysineresidues and the N-terminal amino acid residues; those having a freecarboxyl group may include aspartic acid residues, glutamic acidresidues, and the C-terminal amino acid residue. Sulfhydryl groups, mayalso be used as a reactive group for attaching the polyethylene glycolmolecules. In a preferred embodiment, the polyethylene glycol moleculeis attached at an amino group, such as attachment at the N-terminus orto a lysine side chain amino group.

[0346] As suggested above, polyethylene glycol may be attached topolypeptides via linkage to any of a number of amino acid residues. Forexample, polyethylene glycol can be linked to a polypeptide via covalentbonds to lysine, histidine, aspartic acid, glutamic acid, or cysteineresidues. One or more reaction chemistries may be employed to attachpolyethylene glycol to specific amino acid residues (e.g., lysine,histidine, aspartic acid, glutamic acid, or cysteine) of the polypeptideor to more than one type of amino acid residue (e.g., lysine, histidine,aspartic acid, glutamic acid, cysteine and combinations thereof) of thepolypeptide.

[0347] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration, one may selectfrom a variety of polyethylene glycol molecules (by molecular weight,branching, etc.), the proportion of polyethylene glycol molecules topolypeptide molecules in the reaction mix, the type of pegylationreaction to be performed, and the method of obtaining the selectedN-terminally pegylated polypeptide. The method of obtaining theN-terminally pegylated preparation (i.e., separating this moiety fromother monopegylated moieties if necessary) may be by purification of theN-terminally pegylated material from a population of pegylatedpolypeptide molecules. Selective N-terminal modification of proteins maybe accomplished by reductive alkylation which exploits differentialreactivity of different types of primary amino groups (lysine versus theN-terminus) available for derivatization in a particular protein. Underthe appropriate reaction conditions, substantially selectivederivatization of the protein at the N-terminus with a carbonyl groupcontaining polymer is achieved.

[0348] As indicated above, pegylation of the polypeptides of theinvention may be accomplished by any number of means. For example,polyethylene glycol may be attached to the protein either directly or byan intervening linker. Linkerless systems for attaching polyethyleneglycol to proteins are described in Delgado et al., Crit. Rev. Thera.Drug Carrier Sys., 9:249-304 (1992); Francis et al., Intern. J. ofHematol., 68:1-18 (1998); U.S. Pat. Nos. 4,002,531; 5,349,052; WO95/06058; and WO 98/32466, the disclosures of each of which areincorporated herein by reference.

[0349] One system for attaching polyethylene glycol directly to aminoacid residues of polypeptides without an intervening linker employstresylated MPEG, which is produced by the modification of monomethoxypolyethylene glycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Uponreaction of protein with tresylated MPEG, polyethylene glycol isdirectly attached to amine groups of the polyeptide. Thus, the inventionincludes polypeptide-polyethylene glycol conjugates produced by reactingpolypeptides of the invention with a polyethylene glycol molecule havinga 2,2,2-trifluoreothane sulphonyl group.

[0350] Polyethylene glycol can also be attached to polypeptides using anumber of different intervening linkers. For example, U.S. Pat. No.5,612,460, the entire disclosure of which is incorporated herein byreference, discloses urethane linkers for connecting polyethylene glycolto polypeptides. Polypeptide-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the polypeptide by a linker can alsobe produced by reaction of polypeptides with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichlorophenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber of additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to polypeptides aredescribed in WO 98/32466, the entire disclosure of which is incorporatedherein by reference. Pegylated ACE-2 binding polypeptide productsproduced using the reaction chemistries set out herein are includedwithin the scope of the invention.

[0351] The number of polyethylene glycol moieties attached to eachpolypeptide of the invention (i.e., the degree of substitution) may alsovary. For example, the pegylated polypeptides of the invention may belinked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, ormore polyethylene glycol molecules. Similarly, the average degree ofsubstitution may range within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7,6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18,17-19, or 18-20 polyethylene glycol moieties per polypeptide molecule.Methods for determining the degree of substitution are discussed, forexample, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.,9:249-304 (1992).

[0352] The ACE-2 binding polypeptides of the invention can be recoveredand purified by known methods which include, but are not limited to,ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography (“HPLC”) is employedfor purification.

[0353] The ACE-2 binding polypeptides may also be modified with adetectable label, including, but not limited to, an enzyme, prostheticgroup, fluorescent material, luminescent material, bioluminescentmaterial, radioactive material, positron emitting metal, nonradioactiveparamagnetic metal ion, and affinity label for detection and isolationof ACE-2 target. The detectable substance may be coupled or conjugatedeither directly to the polypeptides of the invention or indirectly,through an intermediate (such as, for example, a linker known in theart) using techniques known in the art. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase,beta-galactosidase, glucose oxidase or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includebiotin, umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin; an example of a luminescent material includes luminol;examples of bioluminescent materials include luciferase, luciferin, andaequorin; and examples of suitable radioactive material include aradioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi,or other radioisotopes such as, for example, iodine (¹³¹I, ¹²⁵I, ¹²³I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In,¹¹³In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti),gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon(¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷R, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn,⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tin.

[0354] In specific embodiments, ACE-2 binding polypetides of theinvention are attached either directly or indirectly, to macrocyclicchelators useful for chelating radiometal ions, including but notlimited to ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. . In apreferred embodiment, the radiometal ion associated with the macrocyclicchelators attached to Neutrokine-alpha and/or Neutrokine-alphaSVpolypeptides of the invention is ¹¹¹In. In another preferred embodiment,the radiometal ion associated with the macrocyclic chelator attached toNeutrokine-alpha and/or Neutrokine-alphaSV polypeptides of the inventionis ⁹⁰Y. In specific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inone embodiment the side chain moiety of one or more classical ornon-classical amino acids in an ACE-2 binding polypeptide comprises aDOTA molecule. In other specific embodiments, the DOTA is attached tothe ACE-2 binding polypeptide of the invention via a linker molecule.Examples of linker molecules useful for conjugating DOTA to apolypeptide are commonly known in the art—see, for example, DeNardo etal., Clin. Cancer Res., 4(10):2483-90 (1998); Peterson et al.,Bioconjug. Chem., 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med.Biol., 26(8):943-50 (1999), which are hereby incorporated by referencein their entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065,which disclose chelating agents that may be conjugated to antibodies,and methods for making and using them, are hereby incorporated byreference in their entireties. Though U.S. Pat. Nos. 5,652,361 and5,756,065 focus on conjugating chelating agents to antibodies, oneskilled in the art could readily adapt the methods disclosed therein inorder to conjugate chelating agents to other polypeptides.

[0355] In a specific embodiment, ACE-2 binding polypeptides of theinvention are labeled with biotin.

[0356] The present invention further encompasses ACE-2 bindingpolypeptides (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants thereof),conjugated to a diagnostic or therapeutic agent. The ACE-2 bindingpolypeptides can be used diagnostically to, for example, monitor orprognose the development or progression of a tumor as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling the ACE-2 bindingpolypeptide to a detectable substance. Examples of detectable substancesinclude, but are not limited to, various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions suchas, for example, those described herein. The detectable substance may becoupled or conjugated either directly to the ACE-2 binding polypeptideor indirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to ACE-2binding polypeptides for use as diagnostics according to the presentinvention.

[0357] Further, an ACE-2 binding polypeptide of the invention (includinga molecule comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof), may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes suchas, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S,⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin,¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium.

[0358] A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include, but are not limited to,paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, thymidine kinase,endonuclease, RNAse, and puromycin and fragments, variants or homologsthereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0359] Techniques known in the art may be applied to label ACE-2 bindingpolypeptides of the invention. Such techniques include, but are notlimited to, the use of bifunctional conjugating agents (see, e.g., U.S.Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931;5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and5,808,003; the contents of each of which are hereby incorporated byreference in its entirety) and direct coupling reactions (e.g.,Bolton-Hunter and Chloramine-T reaction).

[0360] The ACE-2 binding polypeptides of the invention which areconjugates can be used for modifying a given biological response, thetherapeutic agent or drug moiety is not to be construed as limited toclassical chemical therapeutic agents. For example, the drug moiety maybe a protein or polypeptide possessing a desired biological activity.

[0361] An ACE-2 binding polypeptide of the invention (including amolecule comprising, or alternatively consisting of, an ACE-2 bindingpolypeptide fragment or variant thereof), with or without a therapeuticmoiety conjugated to it, administered alone or in combination withcytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.Characterization of ACE-2 Binding Polypeptides

[0362] ACE-2 binding polypeptides of the present invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) may be characterized in avariety of ways. In particular, ACE-2 binding polypeptides and relatedmolecules of the invention may be assayed for the ability tospecifically bind to ACE-2 or a fragment of ACE-2 (e.g., to the solubleform or the membrane-bound form of ACE-2) using techniques describedherein or routinely modifying techniques known in the art. ACE-2 orACE-2 fragments that may be specifically bound by the compositions ofthe invention include, but are not limited to, human ACE-2 (SEQ IDNOs:138, 139, 140 and/or 142) or fragments thereof. Assays for theability of the ACE-2 binding polypeptides of the invention tospecifically bind ACE-2 or a fragment of ACE-2 may be performed insolution (e.g., Houghten, Bio/Techniques, 13:412-421(1992)), on beads(e.g., Lam, Nature, 354:82-84 (1991)), on chips (e.g., Fodor, Nature,364:555-556 (1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), onspores (e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), onplasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA, 89:1865-1869(1992)) or on phage (e.g., Scott and Smith, Science, 249:386-390 (1990);Devlin, Science, 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad.Sci. USA, 87:6378-6382 (1990); and Felici, J. Mol. Biol., 222:301-310(1991)) (each of these references is incorporated herein in its entiretyby reference). ACE-2 binding polypeptides that have been identified tospecifically bind to ACE-2 or a fragment of ACE-2 can then be assayedfor their specificity and affinity for ACE-2 or a fragment of ACE-2using or routinely modifying techniques described herein or otherwiseknown in the art.

[0363] The ACE-2 binding polypeptides of the invention may be assayedfor specific binding to ACE-2 and cross-reactivity with other ACE-2-likepolypeptides by any method known in the art. In particular, the abilityof an ACE-2 binding polypeptide to specifically bind to the soluble formor membrane-bound form of ACE-2 may be determined using or routinelymodifying techniques described herein or otherwise known in art.

[0364] Assays which can be used to analyze specific binding andcross-reactivity include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”assays, “immunoprecipitation” assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, radiometric assays, and fluorescentassays, to name but a few. Such assays are routine and well known in theart (see, e.g., Current Protocols in Molecular Biology, Vol. 1, Ausubelet al, eds. (John Wiley & Sons, Inc., New York 1994), which isincorporated by reference herein in its entirety) and could easily beadapted to make use of an ACE-2 binding polypeptide (possibly inconjunction with an anti-ACE-2 binding polypeptide antibody) in place ofan anti-ACE-2 antibody. Exemplary immunoassays that could be modified touse an ACE-2 binding polypeptide of the invention are described brieflybelow (but are not intended by way of limitation).

[0365] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), incubating themembrane with ACE-2 binding polypeptide (the ACE-2 binding polypeptideof interest) diluted in blocking buffer, washing the membrane in washingbuffer, incubating the membrane with a secondary antibody (whichrecognizes the ACE-2 binding polypeptide) conjugated to an enzyme (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. Alternatively,the ACE-2 binding polypeptide may be directly conjugated to a detectionmolecule (e.g., an enzyme or radiolabel), thereby omitting the need fora secondary anti-ACE-2 binding polypeptide antibody. One of skill in theart would be knowledgeable as to the parameters that can be modified toincrease the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., CurrentProtocols in Molecular Biology, Vol. 1, Ausubel et al, eds. (John Wiley& Sons, Inc., New York 1994) at 10.8.1.

[0366] ELISAs comprise preparing antigen (e.g., ACE-2 target), coatingthe well of a 96-well microtiter plate with the antigen, washing awayantigen that did not bind the wells, adding the ACE-2 bindingpolypeptide of interest conjugated to a detectable compound such as anenzyme (e.g., horseradish peroxidase or alkaline phosphatase) to thewells and incubating for a period of time, washing away unbound ACE-2binding polypeptides or non-specifically bound ACE-2 bindingpolypeptides, and detecting the presence of the ACE-2 bindingpolypeptides specifically bound to the antigen coating the well. InELISAs the ACE-2 binding polypeptide employed in the assay does not haveto be conjugated to a detectable compound; instead, an antibody thatrecognizes the ACE-2 binding polypeptide and that is conjugated to adetectable compound may be added to the well. Further, instead ofcoating the well with the antigen, the ACE-2 binding polypeptide may becoated to the well. In this case, the detectable molecule could be theantigen conjugated to a detectable compound such as an enzyme (e.g.,horseradish peroxidase or alkaline phosphatase). One of skill in the artwould be knowledgeable as to the parameters that can be modified toincrease the signal detected as well as other variations of ELISAs knownin the art. For further discussion regarding ELISAs see, e.g., CurrentProtocols in Molecular Biology, Vol. 1, Ausubel et al, eds. (John Wiley& Sons, Inc., New York 1994) at 11.2.1.

[0367] Immunoprecipitation protocols generally use antibody molecules toimunopreciptate a protein of interest. An ACE-2 preciptation protocolcould easily be modified to use an ACE-2 binding polypeptide in place ofan anti-ACE-2 antibody. Immunopreciptation protocols generally compriselysing a population of cells in a lysis buffer such as RIPA buffer (1%NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented withprotein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,aprotinin, sodium vanadate), adding the antibody of interest to the celllysate, incubating for a period of time (e.g., 1 to 4 hours) at 40degrees C., adding protein A and/or protein G sepharose beads to thecell lysate, incubating for about an hour or more at 40 degrees C.,washing the beads in lysis buffer and resuspending the beads inSDS/sample buffer. If one wanted to substitute an ACE-2 bindingpolypeptide for the anti-ACE-2 antibody one could readily do so, andthen isolate the ACE-2-ACE-2 binding polypeptide complexes with anantibody that recognizes the ACE-2 binding polypeptide. Then the triplecomplex of ACE-2, ACE-2 binding polypeptide, and anti-ACE-2 bindingpolypeptide antibody could be isolated using protein A and/or Protein Gas described above. Such a protocol may be desirable if, for example,the anti-ACE-2 binding polypeptide antibody has a higher affinity forthe ACE-2 binding polypeptide than the anti-ACE-2 antibody may have forACE-2.

[0368] The effectiveness of incorporating an ACE-2 binding polypeptidein an immunoprecipitation protocol to precipitate ACE-2 can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the ACE-2 binding polypeptide to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Current Protocols in Molecular Biology, Vol. 1, Ausubel et al,eds. (John Wiley & Sons, Inc., New York 1994) at 10.16.1.

[0369] The binding affinity of an ACE-2 binding polypeptide (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) to an antigen and theoff-rate of an ACE-2 binding polypeptide-antigen interaction can bedetermined by competitive binding assays. One example of a competitivebinding assay is a modified radioimmunoassay comprising the incubationof labeled antigen (e.g., 3H— or ¹²⁵I-labeled ACE-2 target) with theACE-2 binding polypeptide of interest in the presence of increasingamounts of unlabeled antigen, followed by detection of the ACE-2 bindingpolypeptide bound to the labeled antigen. The affinity of the ACE-2binding polypeptide of the present invention for ACE-2 and the bindingoff-rates can be determined from the data by Scatchard plot analysis.Competition with an anti-ACE-2 antibody or ACE-2 binding polypeptide canalso be determined using radioimmunoassays. In this case, ACE-2 isincubated with an ACE-2 binding polypeptide of the present inventionconjugated to a labeled compound (e.g., with 3H or ¹²⁵I) in the presenceof increasing amounts of an unlabeled ACE-2 binding polypeptide oranti-ACE-2 antibody.

[0370] In a preferred embodiment, BIAcore kinetic analysis is used todetermine the binding on and off rates of ACE-2 binding polypeptides(including molecules comprising, or alternatively consisting of, ACE-2binding polypeptide fragments or variants thereof) to ACE-2, orfragments of ACE-2. BIAcore kinetic analysis comprises analyzing thebinding and dissociation of ACE-2 from chips with immobilized ACE-2binding polypeptides on their surface (see Example 3, infra).

[0371] The ACE-2 binding polypeptides of the invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) can also be assayed for theirability to inhibit, increase, or not significantly alter, the enzymaticactivity of ACE-2 using techniques known to those skilled in the art.For example, cells expressing a substrate for ACE-2 (e.g., angiotensin,bradykinin, tachykinin, neurotensin, Substance P, endothelin, and/orkinetensin) can be contacted with ACE-2 in the presence or absence of anACE-2 binding polypeptide, and the ability of the ACE-2 bindingpolypeptide to inhibit, increase, or not significantly alter, ACE-2binding to the cells can be measured. Alternatively, the ACE-2 bindingpolypeptide may be preincubated with ACE-2 prior to exposure of ACE-2 tocells expressing the ACE-2 receptor. ACE-2 binding to cells can bemeasured by, for example, flow cytometry or a scintillation assay. ACE-2or the ACE-2 binding polypeptide can be labeled with a detectablecompound such as a radioactive label (e.g., ³²P, ³⁵S, and ¹²⁵I) or afluorescent label (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine) to enable detection of an interaction between ACE-2 andits substrates and/or ACE-2 and an ACE-2 binding polypeptide of theinvention.

[0372] The ability of ACE-2 binding polypeptides of the invention toinhibit, increase, or not significantly alter, ACE-2 binding to asubstrate can also be determined in cell-free assays. For example,native or recombinant ACE-2 (e.g., having the amino acid sequence of SEQID NOs:138 and/or 142) or a fragment thereof can be contacted with aACE-2 binding polypeptide and the ability of the ACE-2 bindingpolypeptide to inhibit, increase, or not significantly alter, ACE-2 frombinding to a substrate can be determined. For example, one could use anELISA, or other suitable assay to test the ability of ACE-2 bindingpolypeptides of the invention to inhibit, increase, or not significantlyalter, ACE-2 from binding to a substrate. One way to do such an assaywould be to immobilize the ACE-2 receptor on a solid support. Then ACE-2or ACE-2 fragments labeled with a detectable compound which had beenpreincubated with an ACE-2 binding polypetide of the invention aretested for their ability to bind the ACE-2 substrate immobilized on thesolid support. ACE-2 may be partially or completely purified (e.g.,partially or completely free of other polypeptides) or part of a celllysate. Further, the ACE-2 polypeptide may be a fusion proteincomprising ACE-2 or a biologically active portion thereof and a domainsuch as an Immunoglobulin Fe or glutathione-S-transferase. Additionally,the ACE-2 binding polypeptide and/or ACE-2 substrate may be a fusionprotein comprising an ACE-2 binding portion of the polypeptide substrateand a domain such as an Immunoglobulin Fe orglutathionine-S-transferase. For example, amino acid residues 1-154 ofTACI (GenBank accesion number AAC51790), or 1-48 of BCMA (GenBankaccession number NP_(—)001183) may be fused to the Fe region of an IgGmolecule and used in a cell free assay to determine the ability of ACE-2binding polypeptides of the invention to inhibit, increase, or notsignificantly alter, ACE-2 binding to an ACE-2 substrate. Alternatively,ACE-2 can be biotinylated using techniques well known to those skilledin the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).

[0373] The ACE-2 binding polypeptides of the invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof), can also be assayed fortheir ability to inhibit, stimulate, or not significantly alter,ACE-2-induced enzymatic activity using techniques known to those ofskill in the art. For example, ACE-2 activity can be assayed by³H-thymidine incorporation assays and trypan blue cell counts (see,e.g., Moore et al., Science, 285: 260-263 (1999)). Additionally, theACE-2 binding polypeptides of the invention, or fragments or variantsthereof, can be assayed for their ability to inhibit, stimulate, or notsignificantly alter, ACE-2-induced cleavage of angiotensin usingtechniques known to those of skill in the art (see, e.g., Tipnis et al.,Journal of Biological Chemistry 275:33238-33243(2000)). For example,hydrolysis of angiotensin can be determined by analyzing cleavageproducts detected by high performance liquid chromatography (HPLC).Further, the ACE-2 binding polypeptides of the invention, or fragmentsor varients thereof, can be assayed for their ability to inhibit,stimulate, or not significantly alter ACE-2 regulation of bradykinin,tachykinin, neurotensin, Substance P, and endothelin synthesis and/orcleavage using the same or similar techniques known to those of skill inthe art.

[0374] The ACE-2 binding polypeptides of the invention, or fragments orvariants thereof can also be assayed for their ability to neutralize,enhance, or not significantly alter, ACE-2 activity. For example, ACE-2binding polypeptides or fragments or variants thereof, may be routinelytested for their ability to inhibit ACE-2 from enzymatically acting onany of its substrates (e.g., Angiotensin, bradykinin, tachykinin,neurotensin, Substance P, or endothelin).

[0375] Uses of the Binding Polypeptides and Recombinant Bacteriophage ofthe Invention

[0376] The ACE-2 binding polypeptides described herein are especiallyuseful to detect, isolate, or remove ACE-2 target proteins in solutions.Such solutions may be simple dispersions or solutions of ACE-2 and/orACE-2-like polypeptide in water or aqueous buffer or more complexsolutions, such as, blood and other biological fluids, tissuehomogenates cell extracts, or biopsy samples, and cell culture mediacontaining ACE-2 or ACE-2-like polypeptides. Biological fluids include,but are not limited to sera, plasma, lymph, blood, blood fractionsurine, synovial fluid, spinal fluid, saliva, and mucous.

[0377] In one embodiment, the present invention provides a method fordetecting an ACE-2 protein and/or an ACE-2-like polypeptide in asolution comprising contacting the solution with an ACE-2 bindingpolypeptide of the invention and detecting binding of ACE-2 orACE-2-like polypeptide to the ACE-2 binding polypeptide. The ACE-2binding polypeptide may be either free or immobilized. Preferably, theACE-2 binding polypeptide is a polypeptide immobilized on a solidsurface or chromatographic material or the well of a plastic microtiterassay dish.

[0378] Another embodiment of the present invention is a method forisolating ACE-2 protein and/or ACE-2-like polypeptide from a solutioncontaining it, comprising:

[0379] contacting the solution with an ACE-2 binding polypeptide underconditions that permit binding of ACE-2 and/or ACE-2-like polypeptidesto ACE-2 binding polypeptide, and

[0380] recovering the ACE-2 and/or ACE-2-like polypeptides.

[0381] A further embodiment of the present invention is a method forisolating ACE-2 protein and/or ACE-2-like polypeptide from a solutioncontaining it, comprising:

[0382] contacting the solution with an ACE-2 binding polypeptide underconditions that permit binding of ACE-2 and/or ACE-2-like polypeptidesto ACE-2 binding polypeptide, and

[0383] separating the complex(es) formed by the ACE-2 bindingpolypeptide and ACE-2 and/or ACE-2-like polypeptides from othercomponents of the solution.

[0384] Preferably such method also includes the further steps of:

[0385] dissociating the ACE-2 binding polypeptide from the ACE-2 and/orACE-2-like polypeptides, and

[0386] recovering the dissociated, ACE-2 and/or ACE-2-like polypeptide.

[0387] The invention also provides for kits containing a bindingpolypeptide of the invention for use in methods of detecting orisolating ACE-2 and/or ACE-2-like polypeptides.

[0388] According to the invention, detection or isolation of ACE-2target proteins comprises contacting a solution containing an ACE-2target protein with an ACE-2 binding polypeptide. Depending on theparticular application, the ACE-2 binding polypeptide may be free insolution or immobilized on a solid support or chromatographic material.Sufficient time is allowed to permit binding between the ACE-2 targetprotein and the binding polypeptides, and non-binding components in thesolution or mixture are removed or washed away. The formation of abinding complex between the binding polypeptide and the ACE-2 targetprotein can then be detected, for example, by detecting the signal froma label on the binding polypeptide, which is one component of thebinding complex. A label may be any label that generates a signal thatcan be detected by standard methods, such as a fluorescent label, aradioactive compound, or an enzyme that reacts with a substrate togenerate a detectable signal. Suitable such labels are discussed above.A phage binding polypeptide according to the invention, that is, arecombinant phage displaying an ACE-2 binding polypeptide on itssurface, may form a complex with ACE-2 and/or ACE-2-like polypeptidesthat is detectable as a precipitate or sediment in a reaction tube,which can be detected visually after settling or centrifugation.Alternatively, a sandwich-type assay may be used, wherein an ACE-2binding polypeptide is immobilized on a solid support such as a plastictube or well, or a chromatographic support matrix such as agarose beads,then the solution suspected of containing the ACE-2 target is contactedwith the immobilized binding polypeptide and non-binding materials orcomponents are removed or washed away.

[0389] The binding polypeptides according to this invention areparticularly useful for detection and/or isolation of ACE-2 and/orACE-2-like polypeptides by affinity chromatography methods. Anyconventional method of chromatography may be employed. Preferably, anACE-2 binding polypeptide of the invention will be immobilized on asolid support suitable, for example, for packing a chromatographycolumn. The immobilized ACE-2 binding polypeptide affinity ligand canthen be loaded or contacted with a feed stream under conditionsfavorable to formation of binding polypeptide/ACE-2 (or ACE-2-likepolypeptide) complexes. Non-binding materials can be washed away.Examples of suitable wash conditions can readily be determined by one ofskill in the art and include but are not limited to [PBS/0.01% Tween 20,pH 7.2] and [1M NaCl/10 mm Tris, pH 7.5]. Tris wash buffers may bepreferable since phosphates can preciptate in 50% ethylene glycol. Ingeneral, non-limiting terms, wash buffers are pH 7.0, optionallycontaining 0.0 to 1.5 M NaCl, more preferably 1M NaCl. Additionally,wash buffers may optionally contain a mild detrgenet, such as, forexample, Tween 20, Tween 80, or NP-80. ACE-2 or ACE-2-like polypeptidecan be eluted from the ACE-2 binding polypeptide by introducing solutionconditions that favor dissociation of the binding complex. Suitableelution solutions can readily be determined by one of skill in the artand include but are not limited to [50% ethylene glycol/100 mM NaOAc].By way of non-limiting example, useful elution buffers, for the purposesof the present invention contain 40-60% ethylene glycol, preferably 50%ethylene glycol.; and 50-100 mM NaOAc with a pH in the range of pH 4-pH7, more preferably, pH 4-pH 6 and most preferably pH 4.5-pH 5.5.Preferably, a fast flow affinity chromatographic technique is used tobind the molecules and from which purified ACE-2 or ACE-2-likepolypeptides are eluted.

[0390] Alternatively, batch chromatography can be carried out by mixinga solution containing the ACE-2 target and the ACE-2 bindingpolypeptide, then isolating complexes of the ACE-2 target and thebinding polypeptides. For this type of separation, many methods areknown. For example, the binding polypeptide may be immobilized on asolid support such as beads, then separated from the feed stream alongwith the ACE-2 target by filtration. In another example, the ACE-2binding polypeptide may be modified with its own affinity tag, such as apolyHis tail or streptavidin binding region, which can be used toisolate the binding polypeptide after complexes have formed using animmobilized metal affinity chromatographic resin or steptavidin-coatedsubstrate. Once separated, the ACE-2 target can be released from thebinding polypeptide under elution conditions and recovered in a purifiedform.

[0391] Methods of producing ACE-2 or ACE-2-like polypeptides usuallyyield ACE-2 or ACE-2-like polypeptides in a feed stream thatadditionally contains impurities (with respect to the ACE-2 target). Onepurpose of the present invention is to produce ACE-2 bindingpolypeptides and preparations (such as affinity chromatography media orsurfaces) comprising ACE-2 binding polypeptides that allow rapid andhighly specific purification of ACE-2 target proteins from a feedstream. ACE-2 binding polypeptides obtained herein may easily betailored to isolate ACE-2 target protein from a particular feed stream,using or routinely modifying conditions and techniques known in the art.If an alternate production method for ACE-2 is used, producing adifferent feed stream, a different set of ACE-2 binding polypeptidesand/or conditions may be necessary to achieve the same level ofpurification. The new set of ACE-2 binding polypeptides and/orconditions can be readily obtained following or modifying proceduresoutlined herein, or otherwise known in the art.

[0392] Use of ACE-2 Binding Polypeptides for Epitope Mapping

[0393] The present invention provides ACE-2 binding polypeptides(including molecules comprising, or alternatively consisting of, ACE-2binding polypeptide fragments or variants thereof), that can be used toidentify epitopes of ACE-2. In particular, the ACE-2 bindingpolypeptides of the present invention can be used to identify epitopesof human ACE-2 (SEQ ID NOs:138 and/or 142) or ACE-2 expressed on humanmyocytes and/or proximal tubules and/or epithelial cells usingtechniques described herein or otherwise known in the art. Fragmentswhich function as epitopes may be produced by any conventional means.(See, e.g., Houghten, Proc. Natl. Acad. Sci. USA, 82:5131-5135 (1985),further described in U.S. Pat. No. 4,631,211.)

[0394] Diagnostic Uses of ACE-2 Binding Polypeptides

[0395] Labeled and non-labelled ACE-2 binding polypeptides of theinvention (including molecules comprising, or alternatively consistingof, ACE-2 binding polypeptide fragments or variants thereof) whichspecifically bind to ACE-2 can be used for diagnostic purposes todetect, diagnose, prognose, or monitor diseases and/or disordersassociated with the aberrant expression and/or activity of ACE-2. Theinvention provides for the detection of aberrant expression of ACE-2comprising: (a) assaying the expression of ACE-2 in a biological samplefrom an individual using one or more ACE-2 binding polypeptides of theinvention that specifically binds to ACE-2; and (b) comparing the levelof ACE-2 with a standard level of ACE-2, e.g., in normal biologicalsamples, whereby an increase or decrease in the assayed level of ACE-2compared to the standard level of ACE-2 is indicative of aberrantexpression.

[0396] By “biological sample” is intended any fluids and/or cellsobtained from an individual, body fluid, body tissue, body cell, cellline, tissue culture, or other source which may contain ACE-2 protein ormRNA. Body fluids include, but are not limited to, sera, plasma, urine,synovial fluid, spinal fluid, saliva, and mucous. Tissues samples may betaken from virtually any tissue in the body. Tissue samples may also beobtained from autopsy material. Methods for obtaining tissue biopsiesand body fluids from mammals are well known in the art. Where thebiological sample is to include mRNA, a tissue biopsy is the preferredsource.

[0397] The invention also provides for the detection of aberrantexpression and/or activity of ACE-2 substrates (e.g., angiotensin,bradykinin, tachykinin, neurotensin, Substance P, and endothelin)comprising (a) assaying the expression of ACE-2 substrates in abiological sample from an individual using one or more ACE-2 bindingpolypeptides or fragments or variants thereof that specifically bindsonly to soluble ACE-2, but does not inhibit ACE-2/ ACE-2 substratebinding. Such an ACE-2 binding polypeptide, by way of an example that isnot to be construed as limiting, would be one that is able to capture abiotinylated ACE-2 from solution, but that would not prevent ACE-2 frombinding to it receptor expressed, for example on IM-9 cells, and (b)comparing the level of ACE-2 substrate with a standard level of ACE-2substrate, e.g., in normal tissue or cell samples, whereby an increaseor decrease in the assayed level of ACE-2 substrate compared to thestandard level of ACE-2 substrate is indicative of aberrant expression.

[0398] Angiotensin II is a known potent vasoconstrictor, highconcentrations of which are associated with such diseases ashypertension, congestive heart failure, and several other cardiovasculardisorders. Production of angiotensin II is under control of the ACEenzyme, which competes with ACE-2 for a common substrate, angiotensin.Thus, an an abnomally high agiotensin II level could result fromabnormally low activity of ACE-2, thereby allowing most of all of theangiotensin to be converted to angiotensin II rather than angiotensin1-9. Conversely, inappropriately low concentrations of angiotensin II,which may play a role in hypotensive disorders and shock, may resultfrom inappropriately high activity of ACE-2, whereby most or allangiotensin is converted to angiotensin 1-9 rather than angiotensin II.Furthermore, angiotensin 1-9 potentiates the vasoconstrictor and pressoraction of angiotensin II, indicating a role for angtiotensin 1-9 in theprecise regulation of blood pressure and vascular constriction (seeExample 9).

[0399] Thus, the ACE-2 binding polypeptides of the invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) which specifically bind toACE-2 can be used for diagnostic purposes to detect, diagnose, prognose,or monitor cardiovascular diseases and disorders, including but notlimited to hypertension, hypotension, and/or diseases, disorders, orconditions associated therewith. The invention provides for thedetection of aberrant expression of ACE-2 comprising: (a) assaying theexpression of ACE-2 in a biological sample from an individual using oneor more ACE-2 binding polypeptides of the invention that specificallybinds to ACE-2; and (b) comparing the level of ACE-2 with a standardlevel of ACE-2, e.g., in normal biological samples, whereby an increaseor decrease in the assayed level of ACE-2 compared to the standard levelof ACE-2 is indicative of a cardiovascular disease, disorder, orcondition. In specific embodiments, an increase in the assayed level ofACE-2 is indicative of a cardiovascular disease, disorder, or condition.In other specific embodiments, a decrease in the assayed level of ACE-2is indicative of a cardiovascular disease, disorder, or condition.

[0400] ACE-2 binding polypeptides of the invention (including moleculescomprising, or alternatively consisting of, ACE-2 binding polypeptidefragments or variants thereof) which specifically bind to ACE-2 but donot inhibit ACE-2/ACE-2 substrate binding can be used for diagnosticpurposes to detect, diagnose, prognose, or monitor cardiovasculardiseases and disorders, including but not limited to hypertension and/ordiseases, disorders, or conditions associated therewith. The inventionprovides for the detection of aberrant expression of an ACE-2 substratecomprising: (a) assaying the expression of an ACE-2 substrate in abiological sample from an individual using one or more ACE-2 bindingpolypeptides of the invention that specifically binds to ACE-2; and (b)comparing the level of ACE-2 substrate with a standard level of ACE-2substrate, e.g., in normal biological samples, whereby an increase ordecrease in the assayed level of ACE-2 substrate compared to thestandard level of ACE-2 substrate is indicative of a cardiovasculardisease, disorder, or condition. In specific embodiments, an increase inthe assayed level of ACE-2 substrate is indicative of a cardiovasculardisease, disorder, or condition. In other specific embodiments, adecrease in the assayed level of ACE-2 substrate is indicative of acardiovascular disease, disorder, or condition.

[0401] Cardiac and cardiovascular disorders, diseases, or conditionsthat may be detected, diagnosed, prognosed, or monitored using the ACE-2binding polypeptides of the invention include, but are not limited to,arrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardi al diseases, myocardi alischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

[0402] Additional cardiovascular disorders, diseases, or conditions thatmay be detected, diagnosed, prognosed, or monitored by the ACE-2 bindingpolypeptides of the invention include, but are not limited to,arrhythmias, such as sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0403] Additional cardiovascular disorders, diseases, or conditions thatmay be detected, diagnosed, prognosed, or monitored by the ACE-2 bindingpolypeptides of the invention include, but are not limited to heartvalve diseases, such as aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

[0404] Myocardial diseases that may be detected, diagnosed, prognosed,or monitored, using the ACE-2 binding polypeptides of the inventioninclude, but are not limited to alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[0405] Myocardial ischemias that may be detected, diagnosed, prognosed,or monitored, using the ACE-2 binding polypeptides of the inventioninclude, but are not limited to coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[0406] Additional cardiovascular diseases that may be detected,diagnosed, prognosed, or monitored using the ACE-2 binding polypeptidesof the invention also include vascular diseases such as aneurysms,angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-LindauDisease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome,angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis,Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis,polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies,diabetic retinopathy, embolisms, thrombosis, erythromelalgia,hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension,ischemia, peripheral vascular diseases, phlebitis, pulmonaryveno-occlusive disease, Raynaud's disease, CREST syndrome, retinal veinocclusion, Scimitar syndrome, superior vena cava syndrome,telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

[0407] Aneurysms that may be detected, diagnosed, prognosed, ormonitored using the ACE-2 binding polypeptides of the invention include,but are not limited to dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0408] Arterial occlusive diseases that may be detected, diagnosed,prognosed, or monitored using the ACE-2 binding polypeptides of theinvention include, but are not limited to include, but are not limitedto, arteriosclerosis, intermittent claudication, carotid stenosis,fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoyadisease, renal artery obstruction, retinal artery occlusion, andthromboangiitis obliterans.

[0409] Cerebrovascular disorders that may be detected, diagnosed,prognosed, or monitored using the ACE-2 binding polypeptides of theinvention include, but are not limited to, carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

[0410] Embolisms that may be detected, diagnosed, prognosed, ormonitored using the ACE-2 binding polypeptides of the invention include,but are not limited to air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromoboembolisms. Thrombosis include, but are notlimited to, coronary thrombosis, hepatic vein thrombosis, retinal veinocclusion, carotid artery thrombosis; sinus thrombosis, Wallenberg'ssyndrome, and thrombophlebitis.

[0411] Ischemic disorders that may be detected, diagnosed, prognosed, ormonitored using the ACE-2 binding polypeptides of the invention include,but are not limited to, cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includes,but is not limited to, aortitis, arteritis, Behcet's Syndrome,Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,thromboangiitis obliterans, hypersensitivity vasculitis,Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener'sgranulomatosis.

[0412] In a specific embodiment, the compositions of the presentinvention are used to detect, diagnose, prognose, or monitorhypertension.

[0413] In another specific embodiment, the compositions of the presentinvention are used to detect, diagnose, prognose, or monitor congestiveheart failure.

[0414] In a further specific embodiment, the compositions of the presentinvention are used to detect, diagnose, prognose, or monitorhypotension.

[0415] In an even further specific embodiment, the compositions of thepresent invention are used to detect, diagnose, prognose, or monitorshock.

[0416] Angiotensin II also stimulates the release of aldosterone.Aldosterone is an adrenal cortex hormone that promotes retention of saltand water by the kidneys, which increases plasma volume and, thereby,increases blood pressure. In addition to chronic or acute hypertensionand hypotension, aberrant action of aldosterone is known to causeseveral renal disorders. As discussed previously herein, angiotensin IIconcentration can be correlated with activity or presence of ACE-2.Thus, the ACE-2 binding polypeptides of the invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) which specifically bind toACE-2 can be used for diagnostic purposes to detect, diagnose, prognose,or monitor diseases and disorders associated with aberrant aldosteroneaction, including but not limited to renal diseases and disorders,hypertension, hypotension, and/or diseases, disorders, or conditionsassociated therewith. The invention provides for the detection ofaberrant expression of ACE-2 comprising: (a) assaying the expression ofACE-2 in a biological sample from an individual using one or more ACE-2binding polypeptides of the invention that specifically binds to ACE-2;and (b) comparing the level of ACE-2 with a standard level of ACE-2,e.g., in normal biological samples, whereby an increase or decrease inthe assayed level of ACE-2 compared to the standard level of ACE-2 isindicative of a renal disease, disorder, or condition. In specificembodiments, an increase in the assayed level of ACE-2 is indicative ofa renal disease, disorder, or condition. In other specific embodiments,a decrease in the assayed level of ACE-2 is indicative of a renaldisease, disorder, or condition.

[0417] ACE-2 binding polypeptides of the invention (including moleculescomprising, or alternatively consisting of, ACE-2 binding polypeptidefragments or variants thereof) which specifically bind to ACE-2 but donot inhibit ACE-2/ACE-2 substrate binding can be used for diagnosticpurposes to detect, diagnose, prognose, or monitor diseases anddisorders associated with aberrant aldosterone activity, including butnot limited to renal diseases and/or disorders, hypertension and/ordiseases, disorders, or conditions associated therewith. The inventionprovides for the detection of aberrant expression of an ACE-2 substratecomprising: (a) assaying the expression of an ACE-2 substrate in abiological sample from an individual using one or more ACE-2 bindingpolypeptides of the invention that specifically binds to ACE-2; and (b)comparing the level of ACE-2 substrate with a standard level of ACE-2substrate, e.g., in normal biological samples, whereby an increase ordecrease in the assayed level of ACE-2 substrate compared to thestandard level of ACE-2 substrate is indicative of a renal disease,disorder, or condition. In specific embodiments, an increase in theassayed level of ACE-2 substrate is indicative of a renal disease,disorder, or condition. In other specific embodiments, a decrease in theassayed level of ACE-2 substrate is indicative of a renal disease,disorder, or condition.

[0418] Renal disorders, diseases, and/or conditions that may bedetected, diagnosed, prognosed, monitored, treated, prevented, and/orameliorated using the ACE-2 binding polypeptides of the inventioninclude, but are not limited to acute kidney failure, chronic kidneyfailure, atheroembolic renal failure, end-stage renal disease,inflammatory diseases of the kidney (e.g., acute glomerulonephritis,postinfectious glomerulonephritis, rapidly progressiveglomerulonephritis, nephrotic syndrome, membranous glomerulonephritis,familial nephrotic syndrome, membranoproliferative glomerulonephritis Iand II, mesangial proliferative glomerulonephritis, chronicglomerulonephritis, acute tubulointerstitial nephritis, chronictubulointerstitial nephritis, acute post-streptococcalglomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronicnephritis, interstitial nephritis, and post-streptococcalglomerulonephritis), blood vessel disorders of the kidneys (e.g., kidneyinfarction, atheroembolic kidney disease, cortical necrosis, malignantnephrosclerosis, renal vein thrombosis, renal underperfusion, renalretinopathy, renal ischemia-reperfusion, renal artery embolism, andrenal artery stenosis), and electrolyte imbalances (e.g.,nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria,hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia,hypercalcemia, hypophosphatemia, and hyperphosphatemia).

[0419] In a further embodiment, the ACE-2 binding polypeptides of thepresent invention (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants thereof)which specifically bind to ACE-2 can be used for diagnostic purposes todetect, diagnose, prognose, or monitor diseases and/or disordersassociated with cell proliferation. Smooth muscle cell proliferation inthe intima of muscular arteries is the primary cause of restenosis aftervascular surgery (e.g., angioplasty) and in atherosclerosis. Severalanimal studies have indicated that the renin-angiotensin system plays animportant role in this vascular response. Specifically, it has beenshown that chronic treatment with inhibitors of ACE (e.g., compositionsanalagous to Enalapril, Ramipril, and Captopril) reduces myometrialthickening after balloon injury in rat carotid artery or aorta (Powellet al., Journal of the American College of Cardiology 17: 137B-142B(1991)). Further, it is known that angiotensin II stimulates cell growthand replication in the cardiovascular system through binding angiotensinII receptors (Rosendorff, Journal of the American College of Cardiology28: 803 (1996)). Thus, the compositions of the present invention may beused to detect, diagnose, prognose, or montior diseases and disordersassociated with cell proliferation including, but not limited to,senosis, (e.g., buttonhole stenosis, coronary ostial stenosis, doubleaortic stenosis, fish-mouth mitral stenosis, bronchial stenosis,hypertrophic pyloric stenosis, pyloric stenosis, infundibular stenosis,idiopathic hypertrophic subaortic stenosis, idiopathic subglotticstenosis, pulmonary stenosis, muscular subaortic stenosis, laryngealstenosis, mitral stenosis, supravalvar and subvalvar stenosis,subvalvular and supravalvular stenosis, and tricuspid stenosis),myometrial hypertrophy, hypertrophy or hyperplasia of conduit andresistance vessels, atherosclerosis, and several forms of cancer andneoplastic disorders.

[0420] In a specific embodiment, the present invention encompassesmethods and compositions for detecting, diagnosing and/or prognosingdiseases or disorders of smooth muscle cells.

[0421] In another specific embodiment, the present invention encompassesmethods and compositions for detecting, diagnosing and/or prognosingdiseases or disorders of epithelial cells.

[0422] In further embodiments, the present invention encompasses methodsand compositions for detecting, diagnosing, prognosing and or monitoringgrowth, progression, and/or metastases of malignancies and proliferativediseases or disorders associated with increased cell survival, or theinhibition of apoptosis. For a review of such disorders, see Fishman etal., Medicine, 2d Ed. (J. B. Lippincott Co., Philadelphia 1985).Proliferative diseases and disorders is also extended to includepremalignant conditions (e.g., benign tumors, hyperproliferativedisorders, and benign proliferative disorders—see below) as well asproliferative disorders of smooth muscle cells and endothelial cells.Other abnormal growth conditions that may be treated, diagnosed,prognosed or monitored include, but are not limited to, hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed. (W. B. Saunders Co., Philadelphia 1976), pp. 68-79.)Hyperplasia is a form of controlled cell proliferation involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. As but one example, endometrialhyperplasia often precedes endometrial cancer. Metaplasia is a form ofcontrolled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. Atypicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder.

[0423] In another specific embodiment, the present invention encompassesmethods and compositions for detecting, diagnosing and/or prognosinggrowth, progression, and/or metastases of smooth muscle cells.

[0424] In another specific embodiment, the present invention encompassesmethods and compositions for detecting, diagnosing and/or prognosinggrowth, progression, and/or metastases of epithelial cells.

[0425] As discussed elsewhere herein, bradykinin are believed to also bepeptide substrates of ACE-2. Bradykinin are involved in inflammatoryreactions of various tissues. For example, in the intestine bradykininstimulate contraction of smooth muscle and secretion of ions and fluidin response to injury (Manning et al., Nature 229: 256 (1982)). Thus, ina another embodiment, the ACE-2 binding polypeptides of the presentinvention (including molecules comprising, or alternatively consistingof, ACE-2 binding polypeptide fragments or variants thereof) whichspecifically bind to ACE-2 can be used for diagnostic purposes todetect, diagnose, prognose, or monitor inflammation and diseases and/ordisorders associated therewith. Such conditions include, but are in noway limited to, inflammation associated with infection (e.g., septicshock, sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, acute idiopathic inflammation, alterativeinflammation, atrophic inflammation, catarrhal inflammation, chronic andchronic active inflammation, fibrinopurulent inflammation, graulomatousinflammation, immune inflammation, interstitial inflammation, necroticinflammation, proliferative inflammation, pseudomembranous inflammation,purulent inflammation, serofibrinous inflammation, polytrauma, pain,endotoxin lethality, arthritis (e.g., osteoarthritis and rheumatoidarthritis), complement-mediated hyperacute rejection, nephritis,cytokine or chemokine induced lung injury, inflammatory bowel disease,Crohn's disease, and resulting from over production of cytokines (e.g.,TNF or 1L-1.).

[0426] The invention provides a diagnostic assay for diagnosing orprognosing a disease or disorder, comprising: (a) assaying for the levelof ACE-2 in a biological sample of an individual using one or more ACE-2binding polypeptides of the invention that specifically bind to ACE-2;and (b) comparing the level of ACE-2 with a standard ACE-2 level, e.g.,in a biological sample from a patient without the disease or disorder,whereby an increase or decrease in the assayed ACE-2 level compared tothe standard level of ACE-2 is indicative of a particular disease ordisorder. With respect to cancer, the presence of a relatively highamount of ACE-2 in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0427] ACE-2 binding polypeptides of the invention (including moleculescomprising, or alternatively consisting of, ACE-2 binding polypeptidefragments or variants thereof) can be used to assay protein levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al., J. Cell. Biol., 101:976-985 (1985); Jalkanen et al., J.Cell . Biol., 105:3087-3096 (1987)). Other methods that can be used fordetecting protein gene expression that might utilize ACE-2 bindingpolypeptides or fragments or variants thereof include, but are notlimited to, the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, glucose oxidase, alkalinephophatase, and horseradish peroxidase; radioisotopes, such as iodine(¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹¹¹In, ¹¹²In, ¹¹³In, ^(115m)In), technetium (⁹⁹Tc, ^(99m)Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ^(15f3)Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, 166Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0428] Certain embodiments of the invention are directed to thedetection and diagnosis of a disease or disorder associated withaberrant expression of ACE-2 or ACE-2 substrate in an animal, preferablya mammal and most preferably a human. In one embodiment, diagnosiscomprises: (a) administering (for example, parenterally, subcutaneously,or intraperitoneally) to a subject an effective amount of a labeledACE-2 binding polypeptide of the invention (including moleculescomprising, or alternatively consisting of, ACE-2 binding polypeptidefragments or variants thereof) that specifically binds to ACE-2; (b)waiting for a time interval following the administering for permittingthe labeled ACE-2 binding polypeptide to preferentially concentrate atsites in the subject where ACE-2 is expressed (and for unbound labeledmolecule to be cleared to background level); (c) determining backgroundlevel; and (d) detecting the labeled ACE-2 binding polypeptide in thesubject, such that detection of labeled ACE-2 binding polypeptide orfragment thereof above the background level and above or below the levelobserved in a person without the disease or disorder indicates that thesubject has a particular disease or disorder associated with aberrantexpression of ACE-2 or ACE-2 substrate. Background level can bedetermined by various methods, including comparing the amount of labeledmolecule detected to a standard value previously determined for aparticular system.

[0429] It will be understood by those skilled in the art that the sizeof the subject and the imaging system used will determine the quantityof imaging moiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ⁹⁹Tc. Thelabeled ACE-2 binding polypeptide will then preferentially accumulate atthe location of cells which contain the specific protein. In vivo tumorimaging is described in Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments,” Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0430] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0431] In an embodiment for monitoring of the disease or disorder, themethod is carried out by repeating the method for diagnosing the diseaseor disorder, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc. andcomparing the results of the successive tests.

[0432] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0433] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (see, e.g., Thurston et al., U.S. Pat. No.5,441,050). In another embodiment, the molecule is labeled with afluorescent compound and is detected in the patient using a fluorescenceresponsive scanning instrument. In another embodiment, the molecule islabeled with a positron emitting metal and is detected in the patientusing positron emission-tomography. In yet another embodiment, themolecule is labeled with a paramagnetic label and is detected in apatient using magnetic resonance imaging (MRI).

[0434] Immunophenotyping Using ACE-2 Binding Polypeptides

[0435] The ACE-2 binding polypeptides of the invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) may be utilized forimmunophenotyping of cell lines and biological samples by their ACE-2expression or ACE-2 substrate expression. Various techniques can beemployed utilizing ACE-2 binding polypeptides, fragments, or variants ofthe invention to screen for cellular populations (i.e., cardiacmyocytes, proximal convoluted tubules, endothelial cells, and epithelialcells of Bowman's capsule) expressing ACE-2 or ACE-2 substrate. Suchtechniques include magnetic separation using ACE-2 bindingpolypeptide-coated magnetic beads, “panning” with ACE-2 bindingpolypeptide attached to a solid matrix (i.e., plate), and flow cytometry(see, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell,96:737-49 (1999)). These techniques allow for the screening ofparticular populations of cells.

[0436] In one embodiment, ACE-2 binding polypeptides of the invention(including molecules comprising, or alternatively consisting of, ACE-2binding polypeptide fragments or variants thereof) are used to identifycells, such as cardiac myocytes, proximal convoluted tubules,endothelial cells, and epithelial cells of Bowman's capsule.

[0437] Therapeutic Uses of Peptide Compositions of the Invention

[0438] Co-administration of Angiotensin 1-9 and Angiotensin II had thesurprising result of increasing vasoconstriction to a significantlygreater amount than the sum of the individual peptides administeredalone, suggesting a superadditive or synergistic interaction (seeExample 9). This effect has great therapeutic potential in treatingdiseases and disorders related to inappropriate low blood pressure.Thus, in preferred embodiments for raising blood pressure, peptidecompositions of the invention comprise angiotensin 1-9 and angiotensinII used, for example, in combination with each other, eithersequentially or simultaneously. Thus, the Therapeutic/ProphylacticCompositions and Administration described below for ACE-2 bindingpeptides (e.g., dosage amounts and regimens, routes of adminsitration,and co-administered agents), are applied, in accordance with theinvention, to Angiotensin 1-9 and Angiotensin II as well, in embodimentsrelating to raising blood pressure.

[0439] The present invention is further directed to therapies withinvolve administering angiotensin 1-9/angiotensin 1-9-like polypeptidesin conjunction with angiotensin I/angiotensin II-like polypeptides to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions.

[0440] In a preferred embodiment, angiotensin 1-9/angiotensin 1-9-likepolypeptides in conjunction with angiotensin II/angiotensin II-likepolypeptides is administered to an animal, preferably a mammal, and mostpreferably a human, patient for treating diseases and/or disordersassociated with hypotension, including, for example, shock, syncope, andothers as listed herein.

[0441] The present invention is further directed to ACE-2 bindingpolypeptide-based therapies which involve administering ACE-2 bindingpolypeptides of the invention (including molecules comprising, oralternatively consisting of, ACE-2 binding polypeptide fragments orvariants thereof) to an animal, preferably a mammal, and most preferablya human, patient for treating one or more of the disclosed diseases,disorders, or conditions. Therapeutic compounds of the inventioninclude, but are not limited to, ACE-2 binding polypeptides of theinvention and nucleic acids encoding ACE-2 binding polypeptides of theinvention and antibodies that bind ACE-2 binding polypeptides of theinvention as described herein. The ACE-2 binding polypeptides of theinvention can be used to treat, ameliorate or prevent diseases,disorders or conditions associated with aberrant expression and/oractivity of ACE-2 or ACE-2 substrates, including, but not limited to,any one or more of the diseases, disorders, or conditions describedherein. The treatment and/or prevention of diseases, disorders, orconditions associated with aberrant ACE-2 expression and/or activity oraberrant ACE-2 substrate expression and/or activity includes, but is notlimited to, alleviating symptoms associated with those diseases,disorders or conditions. ACE-2 binding polypeptides of the invention maybe provided in pharmaceutically acceptable compositions as known in theart or as described herein.

[0442] ACE-2 binding polypeptides of the present invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) that function as agonists orantagonists of ACE-2, preferably of ACE-2-induced signal transduction,can be administered to an animal to treat, prevent or ameliorate adisease or disorder associated with aberrant ACE-2 expression, lack ofACE-2 function, aberrant ACE-2 substrate expression, or lack of ACE-2substrate function. For example, ACE-2 binding polypeptides of theinvention which disrupt the interaction between ACE-2 and one or more ofits substrates may be administered to an animal to treat, prevent orameliorate a disease or disorder associated with aberrant ACE-2expression, excessive ACE-2 function, aberrant ACE-2 substrateexpression, or excessive ACE-2 substrate function. ACE-2 bindingpolypeptides of the invention which do not prevent ACE-2 from bindingits substrate but inhibit or downregulate ACE-2-induced signaltransduction can be administered to an animal to treat, prevent orameliorate a disease or disorder associated with aberrant ACE-2expression, excessive ACE-2 function, aberrant ACE-2 substrateexpression, or excessive ACE-2 substrate function. In particular, ACE-2binding polypeptides of the present invention which preventACE-2-induced signal transduction by specifically recognizing theunbound ACE-2, substrate-bound ACE-2, or both unbound andsubstrate-bound ACE-2 can be administered to an animal to treat, preventor ameliorate a disease or disorder associated with aberrant ACE-2expression, excessive ACE-2 function, aberrant ACE-2 substratesexpression, or excessive ACE-2 substrates function.

[0443] The ability of an ACE-2 binding polypeptide of the invention toinhibit or downregulate ACE-2-induced signal transduction may bedetermined by techniques described herein or otherwise known in the art.For example, ACE-2-induced cleavage of ACE-2 substrates can bedetermined by detecting cleavage products via high performance liquidchromatography (HPLC).

[0444] In a specific embodiment, an ACE-2 binding polypeptide of thepresent invention (including molecules comprising, or alternativelyconsisting of, ACE-2 binding polypeptide fragments or variants thereof)that inhibits or reduces ACE-2 activity by at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to ACE-2activity in the absence of the ACE-2 binding polypeptide, isadministered to an animal to treat, prevent or ameliorate a disease ordisorder associated with aberrant ACE-2 expression, excessive ACE-2function, aberrant ACE-2 receptor expression, or excessive ACE-2receptor function. In another embodiment, a combination of ACE-2 bindingpolypeptides, a combination of ACE-2 binding polypeptide fragments, acombination of ACE-2 binding polypeptide variants, or a combination ofACE-2 binding polypeptides, ACE-2 binding polypeptide fragments, and/orvariants that inhibit or reduce ACE-2 activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, at least 60%, at least 55%, at least 50%, at least 45%, at least40%, at least 45%, at least 35%, at least 30%, at least 25%, at least20%, or at least 10% relative to ACE-2 activity in absence of said ACE-2binding polypeptides, ACE-2 binding polypeptide fragments, and/or ACE-2binding polypeptide variants are administered to an animal to treat,prevent or ameliorate a disease or disorder associated with aberrantACE-2 expression, excessive ACE-2 function, aberrant ACE-2 substrateexpression, or excessive ACE-2 substrate function.

[0445] Further, ACE-2 binding polypeptides of the present invention(including molecules comprising, or alternatively consisting of, ACE-2binding polypeptide fragments or variants thereof) which activateACE-2-induced signal transduction can be administered to an animal totreat, prevent or ameliorate a disease or disorder associated withaberrant ACE-2 expression, lack of ACE-2 function, aberrant ACE-2substrate expression, or lack of ACE-2 substrate function. These ACE-2binding polypeptides may potentiate or activate either all or a subsetof the biological activities of ACE-2-mediated substrate action, forexample, by regulating cleavage or synthesis of ACE-2 substrates. TheACE-2 binding polypeptides of the invention may be administered with orwithout being pre-complexed with ACE-2. In a specific embodiment, anACE-2 binding polypeptide of the present invention that increases ACE-2activity by at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99%, or 100% or more relative to ACE-2 activity in absence of theACE-2 binding polypeptide is administered to an animal to treat, preventor ameliorate a disease or disorder associated with aberrant ACE-2expression, lack of ACE-2 function, aberrant ACE-2 substrate expression,or lack of ACE-2 substrate function. In another embodiment, acombination of ACE-2 binding polypeptides, a combination of ACE-2binding polypeptide fragments, a combination of ACE-2 bindingpolypeptide variants, or a combination of ACE-2 binding polypeptides,ACE-2 binding polypeptide fragments and/or ACE-2 binding polypeptidevariants that increase ACE-2 activity by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99%, or 100% or more relative to ACE-2activity in absence of the said ACE-2 binding polypeptides or ACE-2binding polypeptide fragments and/or ACE-2 binding polypeptide variantsis administered to an animal to treat, prevent or ameliorate a diseaseor disorder associated with aberrant ACE-2 expression, lack of ACE-2function, aberrant ACE-2 substrate expression, or lack of ACE-2substrate function.

[0446] In a specific embodiment, the present invention provides a methodof treating, preventing or ameliorating a disease or disorder associatedwith aberrant ACE-2 or ACE-2 substrate expression or activity,comprising administering to an animal in which such treatment,prevention or amelioration is desired, an ACE-2 binding polypeptide inan amount effective to treat, prevent or ameliorate the disease ordisorder. Diseases and disorders which may be treated, prevented orameliorated by this method include, but are not limited to,cardiovascular disorders (e.g., hypertension, chronic heart failure,left ventricular failure, stroke, cerebral vasospasm after subarachnoidinjury, atherosclerotic heart disease, and retinal hemorrhage), renaldisorders (e.g., renal vein thrombosis, kidney infarction, renal arteryembolism, renal artery stenosis, and edema, hydronephritis),proliferative diseases or disorders (e.g., vascular stenosis, myocardialhypertrophy, hypertrophy and/or hyperplasia of conduit and/or resistancevessels, myocyte hypertrophy, and fibroblast proliferative diseases),inflammatory diseases (e.g., SIRS (systemic Inflammatory ResponseSyndromes), sepsis, polytrauma, inflammatory bowl disease, acute andchronic pain, rheumatoid arthritis, and osteo arthritis), allergicdisorders (e.g., asthma, adult respiratory distress syndrome, woundhealing, and scar formation), as well as several other disorders and/ordiseases (e.g., periodontal disease, dysmenorrhea, premature labor,brain edema following focal injury, diffuse axonal injury, andreperfusion injury).

[0447] In a specific embodiment, the present invention provides a methodof treating, preventing or ameliorating diseases or disorders associatedwith hypertension, comprising administering to an animal in which suchtreatment, prevention, or amelioration is desired, an ACE-2 bindingpolypeptide in an amount effective to treat, prevent or ameliorate thedisease or disorder. Diseases and disorders associated with hypertensioninclude, for example, accelerated hypertension, episodic hypertension,paroxysmal hypertension, portal hypertension, primary hypertension,secondary hypertensoin, systemic venous hypertension, borderlinehypertension, adrenal hypertension, benign hypertension, idiopathichypertension, pale hypertension, postpartm hypertension,pregnancy-induced hypertension (gestational hypertension), essentialhypertension, labile hypertension, pulmonary hypertension, renal andrenovascular hypertension, and Goldblatt hypertension, left ventricularfailure, atherosclerotic heart disease, stroke, retinal hemorrhage orinfarction (Keith-Wagener-Barker changes), renal failure, renovasculardisease, chronic heart failure, exudates, papilledema, vascularaccidents, myocardial infarction, dissecting aneurysm

[0448] In a specific embodiment, the present invention provides a methodof treating, preventing or ameliorating diseases or disorders associatedwith hypotension, comprising administering to an animal in which suchtreatment, prevention, or amelioration is desired, an ACE-2 bindingpolypeptide in an amount effective to treat, prevent or ameliorate thedisease or disorder. Diseases and disorders associated with hypotensioninclude, for example, arterial hypotension, idiopathic orthostatichypotension, intracranial hypotension, orthostatic hypotension, inducedor controlled hypotension, shock (e.g., anaphylactic shock,anaphylactiod shock, anestetic shock, cardiogenic shock, chronic shock,deferred or delayed shock, hemorrhagic shock, hypovolemic shock,oligemic shock, septic shock, and vasogenic shock), and syncope (e.g.,local syncope, postural syncope, tussive syncope, and vasodepressorsyncope).

[0449] One or more ACE-2 binding polypeptides of the present invention(including molecules comprising, or alternatively consisting of, ACE-2binding polypeptide fragments or variants thereof) that specificallybind to ACE-2 may be used locally or systemically in the body as atherapeutic. The ACE-2 binding polypeptides of the invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) may also be advantageouslyutilized in combination with monoclonal or chimeric antibodies,lymphokines and/or hematopoietic growth factors (such as, e.g., IL-2,IL-3 and IL-7), for example, which serve to increase the number oractivity of effector cells which interact with the ACE-2 bindingpolypeptides.

[0450] The ACE-2 binding polypeptides of the invention (includingmolecules comprising, or alternatively consisting of, ACE-2 bindingpolypeptide fragments or variants thereof) may be administered alone orin combination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents,anti-angiogenesis and anti-inflammatory agents).

[0451] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing ACE-2 binding polypeptides of theinvention (including molecules comprising, or alternatively consistingof, ACE-2 binding polypeptide fragments or variants thereof) thatspecifically bind to ACE-2, or polynucleotides encoding ACE-2 bindingpolypeptides that specifically bind to ACE-2, for both immunoassaysdirected to and therapy of disorders related to ACE-2 polynucleotides orpolypeptides, including fragments thereof. Such ACE-2 bindingpolypeptides will preferably have an affinity for ACE-2 and/or ACE-2fragments. Preferred binding affinities include those with adissociation constant or K_(D) of less than or equal to 5×10⁻² M, 10⁻²M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵M, or 10 ⁻⁵M. Morepreferably, ACE-2 binding polypeptides of the invention bind ACE-2target proteins with a dissociation constant or K_(D) less than or equalto 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, or 10⁻⁸ M. Even morepreferably, ACE-2 binding polypeptides of the invention bind ACE-2target proteins with a dissociation constant or K_(D) less than or equalto 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

[0452] In a preferred embodiment, ACE-2 binding polypeptides of theinvention neutralize ACE-2 activity. In another preferred embodiment,ACE-2 binding polypeptides of the invention inhibit the activity ofACE-2 substrates, including, for example, angiotensin, bradykinin,tachykinin, neurotensin, Substance P, and endothelin. In a furtherembodiment, the ACE-2 binding polypeptides of the invention areadministered in conjunction with an agent known to inhibit ACE (e.g.,Enalapril®, Captopril®, Fosinopril®, and Pramipril®) to inhibit activityof ACE and/or ACE-2 substrates, including, for example, angiotensin,bradykinin, tachykinin, neurotensin, Substance P, and endothelin.

[0453] In a preferred embodiment, ACE-2 binding polypeptides of theinvention (including molecules comprising, or alternatively consistingof, ACE-2 binding polypeptide fragments or variants thereof) inhibit orreduce binding of the soluble form of ACE-2 to an ACE-2 substrate. Inanother preferred embodiment ACE-2 binding polypeptides of the inventioninhibit or reduce cleavage of ACE-2 substrates induced by the solubleform of ACE-2. In another preferred embodiment ACE-2 bindingpolypeptides of the invention inhibit or reduce the production ofAngiotensin 1-9 and other products of ACE-2 action induced by thesoluble form of ACE-2.

[0454] In a preferred embodiment, ACE-2 binding polypeptides of theinvention (including molecules comprising, or alternatively consistingof, ACE-2 binding polypeptide fragments or variants thereof) inhibit orreduce binding of the membrane-bound form of ACE-2 to an ACE-2substrate. In another preferred embodiment ACE-2 binding polypeptides ofthe invention inhibit or reduce cleavage of ACE-2 substrates induced bythe membrane-bound form of ACE-2. In another preferred embodiment ACE-2binding polypeptides of the invention inhibit or reduce the productionof Angiotensin 1-9 and other products of ACE-2 action induced by themembrane-bound form of ACE-2.

[0455] In a preferred embodiment, ACE-2 binding polypeptides of theinvention (including molecules comprising, or alternatively consistingof, ACE-2 binding polypeptide fragments or variants thereof) inhibit orreduce binding of both the soluble and membrane-bound forms of ACE-2 toan ACE-2 substrate. In another preferred embodiment, ACE-2 bindingpolypeptides of the invention inhibit or reduce cleavage of ACE-2substrates induced by either or both forms of ACE-2. In anotherpreferred embodiment, ACE-2 binding polypeptides of the inventioninhibit or reduce the production of Angiotensin 1-9 and other productsof ACE-2 action induced by either or both forms of ACE-2.

[0456] In one embodiment, the invention provides a method of deliveringradiolabelled ACE-2 binding polypeptide and/or ACE-2 binding polypeptideconjugates of the invention to targeted cells, such as, for example,cardiac myocytes cells expressing the membrane-bound form of ACE-2, orproximal tubules expressing an ACE-2 substrate.

[0457] In one embodiment, the invention provides methods andcompositions for inhibiting or reducing angiotensin 1-9 production,comprising, or alternatively consisting of, contacting an effectiveamount of ACE-2 binding polypeptide with ACE-2, wherein the effectiveamount of ACE-2 binding polypeptide inhibits or reduces ACE-2 mediatedproduction of angiotensin 1-9. In another embodiment, the inventionprovides methods and compositions for inhibiting or reducing angiotensin1-9 production, comprising, or alternatively consisting of,administering to an animal in which such inhibition or reduction isdesired, an ACE-2 binding polypeptide in an amount effective to inhibitor reduce production of angiotensin 1-9.

[0458] Additionally, angiotensin 1-9 can be hydrolyzed by ACE intoangiotensin 1-5. Thus, in another embodiment, the present inventionprovides methods and compositions for inhibiting or reducing angiotensin1-5 production, comprising, or alternatively consisting of, contactingan effective amount of ACE-2 binding polypeptide with ACE-2, wherein theeffective amount of ACE-2 binding polypeptide inhibits or reduces ACE-2mediated production of angiotensin 1-5. In another embodiment, theinvention provides methods and compositions for inhibiting or reducingangiotensin 1-5 production, comprising, or alternatively consisting of,administering to an animal in which such inhibition or reduction isdesired, an ACE-2 binding polypeptide in an amount effective to inhibitor reduce production of angiotensin 1-5.

[0459] Further, ACE-2 and ACE compete for the substrate angiotensin,hydrolyzing angiotensin to angiotensin 1-9 and angiotensin II,respectively. Thus, the present invention provides for methods andcompositions for enhancing or increasing angiotensin II production,comprising, or alternatively consisting of, contacting an effectiveamount of ACE-2 binding polypeptide with ACE-2, wherein the effectiveamount of ACE-2 binding polypeptide enhances or increases ACE mediatedproduction of angiotensin II. In another embodiment, the inventionprovides methods and compositions for enhancing or increasingangiotensin II production, comprising, or alternatively consisting of,administering to an animal in which such enhancing or increasing isdesired, an ACE-2 binding polypeptide in an amount effective to enhanceor increase production of angiotensin II. Determination of angiotenin IIlevels are most often performed by comparing the level of angiotensin IIin a sample to a standard containing a known amount of angiotensin IIusing ELISA assays. Determination of angiotensin II levels in a givensample, can readily be determined using ELISA or other method known inthe art.

[0460] Additionally, ACE-2 has significant sequence homologies with ACEfunctional domains, suggesting that both types of enzymes shareadditional similar substrates beyond angiotensin. Thus, the presentinvention provides for methods and compositions for enhancing orincreasing bradykinin activity, comprising, or alternatively consistingof, contacting an effective amount of ACE-2 binding polypeptide withACE-2, wherein the effective amount of ACE-2 binding polypeptideinhibits or reduces ACE-2 mediated degradation of bradykinin. In anotherembodiment, the invention provides methods and compositions forenhancing or increasing bradykinin activity, comprising, oralternatively consisting of, administering to an animal in which suchenhancing or increasing is desired, an ACE-2 binding polypeptide in anamount effective to enhance or increase activity of bradykinin.Determination of bradykinin levels are most often performed by comparingthe level of bradykinin in a sample to a standard containing a knownamount of bradykinin using ELISA assays. Determination of bradykininlevels in a given sample, can readily be determined using ELISA or othermethod known in the art.

[0461] Additionally, the present invention provides for methods andcompositions for enhancing or increasing tachykinin activity,comprising, or alternatively consisting of, contacting an effectiveamount of ACE-2 binding polypeptide with ACE-2, wherein the effectiveamount of ACE-2 binding polypeptide inhibits or reduces ACE-2 mediateddegradation of tachykinin. In another embodiment, the invention providesmethods and compositions for enhancing or increasing tachykininactivity, comprising, or alternatively consisting of, administering toan animal in which such enhancing or increasing is desired, an ACE-2binding polypeptide in an amount effective to enhance or increaseactivity of tachykinin. Determination of tachykinin levels are mostoften performed by comparing the level of tachykinin in a sample to astandard containing a known amount of tachykinin using ELISA assays.Determination of tachykinin levels in a given sample, can readily bedetermined using ELISA or other method known in the art.

[0462] Additionally, the present invention provides for methods andcompositions for enhancing or increasing neurotensin activity,comprising, or alternatively consisting of, contacting an effectiveamount of ACE-2 binding polypeptide with ACE-2, wherein the effectiveamount of ACE-2 binding polypeptide inhibits or reduces ACE-2 mediateddegradation of neurotensin. In another embodiment, the inventionprovides methods and compositions for enhancing or increasingneurotensin activity, comprising, or alternatively consisting of,administering to an animal in which such enhancing or increasing isdesired, an ACE-2 binding polypeptide in an amount effective to enhanceor increase activity of neurotensin. Determination of neurotensin levelsare most often performed by comparing the level of neurotensin in asample to a standard containing a known amount of neurotensin usingELISA assays. Determination of neurotensin levels in a given sample, canreadily be determined using ELISA or other method known in the art.

[0463] Moreover, the present invention provides for methods andcompositions for enhancing or increasing Substance P activity,comprising, or alternatively consisting of, contacting an effectiveamount of ACE-2 binding polypeptide with ACE-2, wherein the effectiveamount of ACE-2 binding polypeptide inhibits or reduces ACE-2 mediateddegradation of Substance P. In another embodiment, the inventionprovides methods and compositions for enhancing or increasing SubstanceP activity, comprising, or alternatively consisting of, administering toan animal in which such enhancing or increasing is desired, an ACE-2binding polypeptide in an amount effective to enhance or increaseactivity of Substance P. Determination of Substance P levels are mostoften performed by comparing the level of Substance P in a sample to astandard containing a known amount of Substance P using ELISA assays.Determination of Substance P levels in a given sample, can readily bedetermined using ELISA or other method known in the art.

[0464] In addition, the present invention provides for methods andcompositions for enhancing or increasing endothelin activity,comprising, or alternatively consisting of, contacting an effectiveamount of ACE-2 binding polypeptide with ACE-2, wherein the effectiveamount of ACE-2 binding polypeptide inhibits or reduces ACE-2 mediateddegradation of endothelin. In another embodiment, the invention providesmethods and compositions for enhancing or increasing endothelinactivity, comprising, or alternatively consisting of, administering toan animal in which such enhancing or increasing is desired, an ACE-2binding polypeptide in an amount effective to enhance or increaseactivity of endothelin. Determination of endothelin levels are mostoften performed by comparing the level of endothelin in a sample to astandard containing a known amount of endothelin using ELISA assays.Determination of endothelin levels in a given sample, can readily bedetermined using ELISA or other method known in the art.

[0465] Angiotensin, angiotenin II, bradykinin, tachykinin, neurotensin,Substance P, and endothelin all are well known in the art to regulateblood pressure, sodium homeostasis, and inflammatory processes (forreview see Kramer et al., Journal of Cardiovascular Pharmacology 15Suppl. 6: 591-598 (1990); Johnson et al., Journal of HypertensionSupplement 15: S3-S6 (1997); Regoli et al., Regulatory Peptides 45:323-340 (1993); Textor et al., Liver Transplant 6:521-530 (2000)).Errant regulation of blood pressure, sodium homeostasis, or inflammatoryresponses affects several physiological systems, including thecardiovascular system, renal system, and the immune system. Bymodulating activity of angiotensin, angiotenin II, bradykinin,tachykinin, neurotensin, Substance P, and endothelin, compositions ofthe present invention can be used as therapeutic or pharmaceutical agentto treat, prevent, or ameliorate diseases and/or disorders associatedwith aberrant blood pressure, sodium homeostasis, or inflammatoryprocesses.

[0466] In one embodiment, therapeutic or pharmaceutical compositions ofthe present invention are administered to an animal to treat, prevent,or ameliorate diseases and/or disorders associated with hypertensionincluding, but not limited to, accelerated hypertension, episodichypertension, paroxysmal hypertension, portal hypertension, primaryhypertension, secondary hypertensoin, systemic venous hypertension,borderline hypertension, adrenal hypertension, benign hypertension,idiopathic hypertension, pale hypertension, postpartm hypertension,pregnancy-induced hypertension (gestational hypertension), essentialhypertension, labile hypertension, pulmonary hypertension, renal andrenovascular hypertension, and Goldblatt hypertension, left ventricularfailure, atherosclerotic heart disease, stroke, retinal hemorrhage orinfarction (Keith-Wagener-Barker changes), renal failure, renovasculardisease, exudates, papilledema, vascular accidents, myocardialinfarction, dissecting aneurysm.

[0467] In another embodiment, therapeutic or pharmaceutical compositionsof the present invention are administered to an animal to treat,prevent, or ameliorate diseases and/or disorders associated withhypotension including, but not limited to, arterial hypotension,idiopathic orthostatic hypotension, induced or controlled hypotension,shock (e.g., anaphylactic shock, anaphylactoid shock, anestetic shock,cardiogenic shock, chronic shock, deferred or delayed shock, hemorrhagicshock, hypovolemic shock, oligemic shock, septic shock, and vasogenicshock), and syncope (e.g., local syncope, postural syncope, tussivesyncope, and vasodepressor syncope).

[0468] In a preferred embodiment, therapeutic compositions of thepresent invention are administered to an animal (preferrablyadministered to a human) to treat, prevent, or ameliorate diseasesand/or disorders associated with shock.

[0469] In a further preferred embodiment, shock is treated, prevented,or ameliorated by administering to an animal (preferrably administeredto a human) angiotensin 1-9/ angiotensin 1-9-like polypeptides inconjunction with angiotensin II/angiotensin II-like polypeptides (seeExample 9).

[0470] In another preferred embodiment, shock is treated, prevented, orameliorated by administering to an animal (preferrably administered to ahuman) compositions of the invention that increase concentrations ofangiotensin 1-9/angiotensin 1-9-like polypeptides and/or angiotensinI/angiotensin II-like polypeptides.

[0471] In another embodiment, therapeutic or pharmaceutical compositionsof the present invention are administered to an animal to treat,prevent, or ameliorate diseases and/or disorders associated withcardiovascular disease including, but not limited to, arrhythmias (e.g.,sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia,extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrialblock, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome,Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome,sick sinus syndrome, tachycardias (e.g., paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia),and ventricular fibrillation), carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure,congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, hearthypertrophy, congestive cardiomyopathy, left ventricular hypertrophy,right ventricular hypertrophy, post-infarction heart rupture,ventricular septal rupture, heart valve diseases (e.g., aortic valveinsufficiency, aortic valve stenosis, hear murmurs, aortic valveprolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valveinsufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valveinsufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspidvalve insufficiency, and tricuspid valve stenosis), myocardial diseases(e.g., alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophiccardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvularstenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardialfibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardialreperfusion injury, and myocarditis), myocardial ischemia (e.g.,coronary disease, such as angina pectoris, coronary aneurysm, coronaryarteriosclerosis, coronary thrombosis, coronary vasospasm, myocardialinfarction and myocardial stunning), pericardial effusion, pericarditis(including constrictive and tuberculous), pneumopericardium,postpericardiotomy syndrome, pulmonary heart disease, rheumatic heartdisease, ventricular dysfunction, hyperemia, cardiovascular pregnancycomplications, Scimitar Syndrome, cardiovascular syphilis,cardiovascular tuberculosis, aneurysms (e.g., dissecting aneurysms,false aneurysms, infected aneurysms, ruptured aneurysms, aorticaneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, andiliac aneurysms), angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases (e.g.,arteriosclerosis, intermittent claudication, carotid stenosis,fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoyadisease, renal artery obstruction, retinal artery occlusion, andthromboangiitis obliterans), arteritis, enarteritis, polyarteritisnodosa, cerebrovascular disorders (e.g., carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency), diabetic angiopathies, diabetic retinopathy, embolisms(e.g., air embolisms, amniotic fluid embolisms, cholesterol embolisms,blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms), thrombosis (e.g., coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis),erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia (e.g., cerebral ischemia, ischemiccolitis, compartment syndromes, anterior compartment syndrome,myocardial ischemia, reperfusion injuries, and peripheral limbischemia), peripheral vascular diseases, phlebitis, pulmonaryveno-occlusive disease, Raynaud's disease, CREST syndrome, retinal veinocclusion, Scimitar syndrome, superior vena cava syndrome,telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis(e.g., aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis), and venousinsufficiency.

[0472] In a further embodiment, therapeutic or pharmaceuticalcompositions of the present invention are administered to an animal totreat, prevent, or ameliorate diseases and/or disorders associated withthe renal system including, but not limited to, acute kidney failure,chronic kidney failure, atheroembolic renal failure, end-stage renaldisease, inflammatory diseases of the kidney (e.g., acuteglomerulonephritis, postinfectious glomerulonephritis, rapidlyprogressive glomerulonephritis, nephrotic syndrome, membranousglomerulonephritis, familial nephrotic syndrome, membranoproliferativeglomerulonephritis I and II, mesangial proliferative glomerulonephritis,chronic glomerulonephritis, acute tubulointerstitial nephritis, chronictubulointerstitial nephritis, acute post-streptococcalglomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronicnephritis, interstitial nephritis, and post-streptococcalglomerulonephritis), blood vessel disorders of the kidneys (e.g., kidneyinfarction, atheroembolic kidney disease, cortical necrosis, malignantnephrosclerosis, renal vein thrombosis, renal underperfusion, renalretinopathy, renal ischemia-reperfusion, renal artery embolism, andrenal artery stenosis), and electrolyte imbalances (e.g.,nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria,hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia,hypercalcemia, hypophosphatemia, and hyperphosphatemia).

[0473] In an even further embodiment, therapeutic or pharmaceuticalcompositions of the present invention are administered to an animal totreat, prevent, or ameliorate diseases and/or disorders associated withinflammatory responses. Such inflammatory conditions include, but arenot limited to, for example, respiratory disorders (such as, e.g.,asthma and allergy); gastrointestinal disorders (such as, e.g.,inflammatory bowel disease); cancers (such as, e.g., gastric, ovarian,lung, bladder, liver, and breast); CNS disorders (such as, e.g.,multiple sclerosis, blood-brain barrier permeability, ischemic braininjury and/or stroke, traumatic brain injury, neurodegenerativedisorders (such as, e.g., Parkinson's disease and Alzheimer's disease),AIDS-related dementia, and prion disease); cardiovascular disorders(such as, e.g., atherosclerosis, myocarditis, cardiovascular disease,and cardiopulmonary bypass complications); as well as many additionaldiseases, conditions, and disorders that are characterized byinflammation (such as, e.g., chronic hepatitis (B and C), rheumatoidarthritis, gout, trauma, septic shock, pancreatitis, sarcoidosis,dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemiclupus erythematosis, diabetes mellitus (i.e., type 1 diabetes), andallogenic transplant rejection).

[0474] Similarly, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention may also be used totreat, prevent, or ameliorate inflammation, including, but not limitedto, inflammation associated with infection (e.g., septic shock, sepsis,or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, polytrauma, pain, endotoxin lethality, arthritis (e.g.,osteoarthritis and rheumatoid arthritis), complement-mediated hyperacuterejection, nephritis, cytokine or chemokine induced lung injury,inflammatory bowel disease, Crohn's disease, and resulting from overproduction of cytokines (e.g., TNF or IL-1.).

[0475] Additionally, many autoimmune disorders are in part manifested asinappropriate inflammation resulting from inappropriate recognition ofself as foreign material by immune cells. Therefore, the administrationof polynucleotides and polypeptides of the invention that can inhibit aninappropriate inflammatory response may be an effective therapy inpreventing autoimmune disorders.

[0476] Autoimmune diseases and/or disorders that may be treated,prevented, or ameliorated by compositions of the present inventioninclude, but not limited to, autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgAnephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation,Autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitis,and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism(i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus,Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, forexample, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulinresistance, autoimmune hemolytic anemia, autoimmune thrombocytopenicpurpura, rheumatoid arthritis, schleroderma with anti-collagenantibodies, mixed connective tissue disease,polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison'sdisease, infertility, glomerulonephritis such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren'ssyndrome, diabetes millitus, and adrenergic drug resistance (includingadrenergic drug resistance with asthma or cystic fibrosis), chronicactive hepatitis, primary biliary cirrhosis, other endocrine glandfailure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria,atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulamatous, degenerative, and atrophic disorders.

[0477] Similarly, in specific embodiments, polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, are useful to treat, diagnose, and/or prevent transplantationrejections, graft-versus-host disease, autoimmune and inflammatorydiseases (e.g., immune complex-induced vasculitis, glomerulonephritis,hemolytic anemia, myasthenia gravis, type II collagen-induced arthritis,experimental allergic and hyperacute xenograft rejection, rheumatoidarthritis, and systemic lupus erythematosus (SLE). Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. Polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, that inhibit an immune response, particularly the activation,proliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing organ rejection or GVHD.

[0478] Further, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed using polypeptides, antibodies,or polynucleotides of the invention, and/or agonists or antagoniststhereof. Moreover, these molecules can be used to treat, prevent, and/ordiagnose anaphylaxis, hypersensitivity to an antigenic molecule, orblood group incompatibility.

[0479] Beyond its role as a neuromodulator, neurotensin is found in highconcentrations in gut endocrine cells of the ileum and is releasedfollowing ingestion of food. Since ACE-2 has been found to hydrolyzeneurotensin (as described herein), the invention provides for methodsand compositions that can be used for digestive purposes.

[0480] Thus, in one embodiment, the invention can be used to treat,prevent, or ameliorate diseases and disorders of the digestive systemincluding, but not limited to, disorders of the small intestine, such asmalabsorption syndromes, distension, irritable bowel syndrome, sugarintolerance, celiac disease, duodenal ulcers, duodenitis, tropicalsprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease,appendicitis, obstructions of the ileum, Meckel's diverticulum, multiplediverticula, failure of complete rotation of the small and largeintestine, lymphoma, and bacterial and parasitic diseases (such asTraveler's diarrhea, typhoid and paratyphoid, cholera, infection byRoundworms (Ascariasis lumbricoides), Hookworms (Ancylostoma duodenale),Threadworms (Enterobius vermicularis), Tapeworms (Taenia saginata,Echinococcus granulosus, Diphyllobothrium spp., and T. solium).

[0481] The invention can also be used to treat, prevent, or amelioratediseases and disorders disorders of the large intestine, includingantibiotic-associated colitis, diverticulitis, ulcerative colitis,acquired megacolon, abscesses, fungal and bacterial infections,anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps(e.g., villous adenoma), colon carcinoma, colorectal cancer], colonicdiverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease,toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]),constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery),duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenalulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ilealdiseases (ileal neoplasms, ileitis), immunoproliferative smallintestinal disease, inflammatory bowel disease (ulcerative colitis,Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis,balantidiasis, blastocystis infections, cryptosporidiosis,dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula(rectal fistula), intestinal neoplasms (cecal neoplasms, colonicneoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps,jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferentloop syndrome, duodenal obstruction, impacted feces, intestinalpseudo-obstruction [cecal volvulus], intussusception), intestinalperforation, intestinal polyps (colonic polyps, gardner syndrome,peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms),malabsorption syndromes (blind loop syndrome, celiac disease, lactoseintolerance, short bowl syndrome, tropical sprue, whipple's disease),mesenteric vascular occlusion, pneumatosis cystoides intestinalis,protein-losing enteropathies (intestinal lymphagiectasis), rectaldiseases (anus diseases, fecal incontinence, hemorrhoids, proctitis,rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenalulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer,Zollinger-Ellison syndrome), postgastrectomy syndromes (dumpingsyndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux(bile reflux), gastric antral vascular ectasia, gastric fistula, gastricoutlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis,stomach dilatation, stomach diverticulum, stomach neoplasms (gastriccancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastricpolyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis,visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum,postoperative nausea and vomiting) and hemorrhagic colitis.

[0482] Additionally, the invention can be used to treat, prevent, orameliorate diseases and disorders disorders of the liver, such asintrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis),fatty liver (alcoholic fatty liver, reye syndrome), hepatic veinthrombosis, hepatolentricular degeneration, hepatomegaly,hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension(esophageal and gastric varices), liver abscess (amebic liver abscess),liver cirrhosis (alcoholic, biliary and experimental), alcoholic liverdiseases (fatty liver, hepatitis, cirrhosis), parasitic (hepaticechinococcosis, fascioliasis, amebic liver abscess), jaundice(hemolytic, hepatocellular, and cholestatic), cholestasis, portalhypertension, liver enlargement, ascites, hepatitis (alcoholichepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B,hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral humanhepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitisE), Wilson's disease, granulomatous hepatitis, secondary biliarycirrhosis, hepatic encephalopathy, portal hypertension, varices, hepaticencephalopathy, primary biliary cirrhosis, primary sclerosingcholangitis, hepatocellular adenoma, hemangiomas, bile stones, liverfailure (hepatic encephalopathy, acute liver failure), and liverneoplasms (angiomyolipoma, calcified liver metastases, cystic livermetastases, epithelial tumors, fibrolamellar hepatocarcinoma, focalnodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma,hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liverhemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors ofliver, nodular regenerative hyperplasia, benign liver tumors (Hepaticcysts [Simple cysts, Polycystic liver disease, Hepatobiliarycystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymalhamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis,Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors[Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma),Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerativehyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma,hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma,cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi'ssarcoma, hemangioendothelioma, other tumors, embryonal sarcoma,fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosishepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittentporphyria, porphyria cutanea tarda), Zellweger syndrome).

[0483] Moreover, the invention can be used to treat, prevent, orameliorate diseases and disorders disorders of the gallbladder, such asgallstones (cholelithiasis and choledocholithiasis), postcholecystectomysyndrome, diverticulosis of the gallbladder, acute cholecystitis,chronic cholecystitis, bile duct tumors, and mucocele.

[0484] Further, the invention can be used to treat, prevent, orameliorate diseases and disorders disorders of the pancreas including,but not limited to, acute pancreatitis, chronic pancreatitis (acutenecrotizing pancreatitis, alcoholic pancreatitis), neoplasms(adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma,gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors,pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis,cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).

[0485] Other diseases and disoders of the gastroinstestinal system thatcan be treated, prevented, or ameliorated by compositions of the presentinvention include dysphagia, odynophagia, inflammation of the esophagus,peptic esophagitis, gastric reflux, submucosal fibrosis and stricturing,Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers,adeoncarcinomas, gastric retention disorders, gastroenteritis, gastricatrophy, gastric/stomach cancers, polyps of the stomach, autoimmunedisorders such as pernicious anemia, pyloric stenosis, gastritis(bacterial, viral, eosinophilic, stress-induced, chronic erosive,atrophic, plasma cell, and Ménétrier's), peritoneal diseases (e.g.,chyloperioneum, hemoperitoneum, mesenteric cyst, mesentericlymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms,peritonitis, pneumoperitoneum, bubphrenic abscess,), biliary tractdiseases, such as, gastroschisis, fistula (e.g., biliary fistula,esophageal fistula, gastric fistula, intestinal fistula, pancreaticfistula), neoplasms (e.g., biliary tract neoplasms, esophagealneoplasms, such as adenocarcinoma of the esophagus, esophageal squamouscell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, suchas adenocarcinoma of the pancreas, mucinous cystic neoplasm of thepancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritonealneoplasms), esophageal disease (e.g., bullous diseases, candidiasis,glycogenic acanthosis, ulceration, barrett esophagus varices, atresia,cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g.,tracheoesophageal fistula), motility disorders (e.g., CREST syndrome,deglutition disorders, achalasia, spasm, gastroesophageal reflux),neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weisssyndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatalhernia); gastrointestinal diseases, such as, gastroenteritis (e.g.,cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis,melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer,gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g.,congenital diaphragmatic hernia, femoral hernia, inguinal hernia,obturator hernia, umbilical hernia, ventral hernia), and intestinaldiseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).

[0486] Several in vivo studies have indicated that the renin-angiotensinsystem plays an important role in the proliferation of smooth musclecells. For example, recent evidence indicates that Angiotensin IIstimulates the growth of vascular smooth muscle cells in response toinjury (see Inagami and Eguchi, Brazilian Journal of Medical andBiological Research 33: 619-624 (2000)). Specifically, endogenousangiotensin II stimulates the progression from GI to S phase in vascularsmooth muscle cells (Kubo et al., American Journal of Hypertension,13:1117-1124 (2000)). This poses a particular problem relative to manysurgical procedures, such as angioplasty, where smooth muscle cellproliferation is a primary cause of artery restenosis.

[0487] ACE-2 directly competes with ACE for angiotensin to produceangiotensin 1-9 and angiotensin II, respectively. Thus, it is logical toassume that regulation of ACE-2 activity affects production ofangiotensin II. Specifically, inhibition of ACE-2 prevents theconversion of angiotenin to angiotensin 1-9, making available anincreased concentration of angiotenin for conversion to angiotensin II.Conversely, stimulation of ACE-2 increases utlization of angiotensin,decreasing the amount available for conversion to angiotensin II. Thus,the invention provides methods and compositions for regulatingangiotenin II-mediated cell proliferation.

[0488] Hence, in another embodiment, therapeutic or pharmaceuticalcompositions of the present invention are administered to an animal totreat, prevent, or ameliorate diseases and/or disorders associated withcell proliferation including, but not limited to, neoplasms located inthe: colon, abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital tissues; and lymphoproliferative disorders,paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron'sMacroglobulinemia, Gaucher's Disease, histiocytosis, and any otherhyperproliferative disease, besides neoplasia, located in anaforementioned organ system.

[0489] In a preferred embodiment, the compositions of the presentinvention can be used to treat, prevent, or ameliorate stenosis,restenosis, myocardial hypertrophy; hypertrophy or hyperplasia ofconduit and resistance vessels, and atherosclerosis.

[0490] Pain and hyperalgesia, the perceptual companions of tissue injuryand inflammation, are in part attributable to the sensitization ofprimary afferent nociceptors by endogenously released chemicals, such asbradykinin. Application of exogenous bradykinin or stimulation ofendogenous bradykinin production has been demonstrated to causehyperalgesia in animal models of pain (for review see Burch and Kyle,Life Sciences 50: 829-838 (1992)). Additionally, a number of studieshave shown that bradykinin antagonists are capable of blocking orameliorating pain in both animal models and humans (for review see Burchet al., Medical Research Review 10: 237-269 (1990); Sharma, GeneticPharmacology 24: 267-274 (1993)). As discussed elsewhere herein, thecompositions of the invention may be used to regulate the hydrolysis(and, therefore, activity) of bradykinin. Thus, the invention providesmethods and compositions for regulating bradykinin-mediated pain andhyperalgesia.

[0491] In one embodiment, therapeutic or pharmaceutical compositions ofthe present invention can be used as analgesics to reduce or inhibitpain. For example, therapeutic or pharmaceutical compositions of thepresent invention can be used to treat, prevent, or ameliorateperioperative pain and/or for use in surgical procedures and labor.

[0492] Additionally, therapeutic or pharmaceutical compositions of thepresent invention can be used to treat, prevent, or ameliorate painassociated with cancer and treatment of cancer (e.g., radiation therapy,surgery, and/or chemotherapy).

[0493] Moreover, therapeutic or pharmaceutical compositions of thepresent invention can be used to treat, prevent, or ameliorateneuropathic pain disorders including, but not limited to, reflexsympathetic dystrophy, causalgia, phantom limb pain, trigeminalneuralgia, a typical trigeminal neuralgia, geniculate neuralgia,glossopharyngeal neuralgia, hallucinatory neuralgia, idiopathicneuralgia, intercostal neuralgia, mammary neuralgia, Morton neuralgia,occipital neuralgia, periodic migrainous neuralgia, sciatic neuralgia,sphenopalatine neuralgia, suboccipital neuralgia, supraorbitalneuralgia, and symptomatic neuralgia.

[0494] Therapeutic or pharmaceutical compositions of the presentinvention can also be used to treat, prevent, or ameliorate idiopathicpain.

[0495] Additionally, therapeutic or pharmaceutical compositions of thepresent invention can be used to treat, prevent, or ameliorate painassociated with diseases and/or disorders including, but in no waylimited to, burns, angina, myocardial ischemia, minor or severe trauma,migraine, shock, arthritis, rheumatoid arthritis, infection (e.g.,herpes zoster, AIDS and AIDS-related conditions, sepsis, and pneumonia),and rhinitis.

[0496] Further, therapeutic or pharmaceutical compositions of thepresent invention can be used to treat pain of the body including, forexample, abdominal pain, back pain (particularly lower back pain),pelvic pain, joint pain, headache, facial pain, and muscular pain, aswell as bodily pain due to trauma, stings, bites, and central nervoussystem injury.

[0497] In another embodiment, the presence of ACE-2 in the testissuggests that the present invention may also have utility in treatinginfertility or other disorders relating to male reproduction (e.g.,erectile dysfunction) and/or gamete formation and maturation.

[0498] In a further embodiment, compositions of the present inventioncan be useful in treating, preventing, or ameliorating cognitivediseases.

[0499] In another embodiment, the invention provides a method for thespecific delivery of ACE-2 binding polypeptides and ACE-2 bindingpolypeptide conjugates of the invention to cells by administeringmolecules of the invention that are associated with heterologouspolypeptides or nucleic acids. In one example, the invention providesfor a method for delivering a therapeutic protein into the targetedcell. In another example, the invention provides a method for deliveringa single strand nucleic acid (e.g., antisense or ribozymes) or doublestranded nucleic acid (e.g., DNA that can integrate into the cell'sgenome or replicate episomally and that can be transcribed) in thetarget cell.

[0500] Gene Therapy

[0501] In a specific embodiment, nucleic acids comprising sequencesencoding ACE-2 binding polypeptides or functional derivatives thereof,are administered to treat, inhibit or prevent a disease or disorderassociated with aberrant expression and/or activity of ACE-2 and/or itssubstrates, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded protein that mediates a therapeuticeffect.

[0502] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0503] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy, 12:488-505 (1993); Wu and Wu, Biotherapy,3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol., 32:573-596(1993); Mulligan, Science, 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem., 62:191-217 (1993); May, TIBTECH, 1 1(5):155-215(1993). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Current Protocols in MolecularBiology, Ausubel et al., eds. (John Wiley & Sons, NY 1993); andKriegler, Gene Transfer and Expression, A Laboratory Manual (StocktonPress, NY 1990).

[0504] In a preferred aspect, a composition of the invention comprises,or alternatively consists of, nucleic acids encoding an ACE-2 bindingpolypeptide, said nucleic acids being part of an expression vector thatexpresses the ACE-2 binding polypeptide or fragment thereof or chimericprotein including it in a suitable host. In particular, such nucleicacids have promoters, preferably heterologous promoters, operably linkedto the ACE-2 binding polypeptide coding region, said promoter beinginducible or constitutive, and, optionally, tissue-specific. In anotherparticular embodiment, nucleic acid molecules are used in which theACE-2 binding polypeptide coding sequences and any other desiredsequences are flanked by regions that promote homologous recombinationat a desired site in the genome, thus providing for intrachromosomalexpression of the ACE-2 binding polypeptide encoding nucleic acids(Koller and Smithies, Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989);Zijlstra et al., Nature, 342:435-438 (1989).

[0505] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0506] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT publications WO 92/06180; WO 92/22635; WO92/20316; WO 93/14188, WO 93/20221). Alternatively, the nucleic acid canbe introduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA, 86:8932-8935 (1989); Zijlstra et al., Nature,342:435-438 (1989)).

[0507] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an ACE-2 binding polypeptide of the invention orfragments or variants thereof are used. For example, a retroviral vectorcan be used (see Miller et al., Meth. Enzymol., 217:581-599 (1993)).These retroviral vectors contain the components necessary for thecorrect packaging of the viral genome and integration into the host cellDNA. The nucleic acid sequences encoding the ACE-2 binding polypeptideto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. Additional detailsconcerning retroviral vectors can be found in Boesen et al., Biotherapy,6:29 1-302 (1994), which describes the use of a retroviral vector todeliver the mdr 1 gene to hematopoietic stem cells in order to make thestem cells more resistant to chemotherapy. Other references illustratingthe use of retroviral vectors in gene therapy are: Clowes et al., J.Clin. Invest., 93:644-651(1994); Klein et al., Blood, 83:1467-1473(1994); Salmons and Gunzberg, Human Gene Therapy, 4:129-141 (1993); andGrossman and Wilson, Curr. Opin. in Genetics and Devel., 3:110-114(1993).

[0508] Other viral vectors that can be used in gene therapy areadenoviruses. Adenoviruses are especially attractive vehicles fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia, where they cause a mild disease. Other targetsfor adenovirus-based delivery systems are liver, the central nervoussystem, endothelial cells, and muscle. Adenoviruses have the advantageof being capable of infecting non-dividing cells. See, Kozarsky andWilson, Current Opinion in Genetics and Development, 3:499-503 (1993),presenting a review of adenovirus-based gene therapy. Bout et al., HumanGene Therapy, 5:3-10 (1994) demonstrated the use of adenovirus vectorsto transfer genes to the respiratory epithelia of rhesus monkeys. Otherinstances of the use of adenoviruses in gene therapy can be found inRosenfeld et al., Science, 252:431-434 (1991); Rosenfeld et al., Cell,68:143-155 (1992); Mastrangeli et al., J. Clin. Invest., 91:225-234(1993); PCT publication WO 94/12649; and Wang et al., Gene Therapy,2:775-783 (1995). In a preferred embodiment, adenovirus vectors areused.

[0509] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med., 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0510] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0511] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.,217:599-618 (1993); Cohen et al., Meth. Enzymol., 217:618-644 (1993);Clin. Pharma. Ther., 29:69-92m (1985)) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0512] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0513] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to cardiac myocytes, proximal tubules, endothelialcells, epithelial cells, keratinocytes, fibroblasts, muscle cells,hepatocytes; blood cells such as T lymphocytes, B lymphocytes,monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,granulocytes; various stem or progenitor cells, in particularhematopoietic stem or progenitor cells, e.g., as obtained from bonemarrow, umbilical cord blood, peripheral blood, fetal liver, etc.

[0514] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0515] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an ACE-2 binding polypeptide orfragment thereof are introduced into the cells such that they areexpressible by the cells or their progeny, and the recombinant cells arethen administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells that can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention (see, e.g., PCT publication WO 94/08598; Stemple and Anderson,Cell, 7 1:973-985 (1992); Rheinwald, Meth. Cell Bio., 21A:229 (1980);and Pittelkow and Scott, Mayo Clinic Proc., 61:771 (1986)).

[0516] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription.

[0517] Demonstration of Therapeutic or Prophylactic Utility of aComposition

[0518] The compounds of the invention are preferably tested in vitro,and then in vivo for the desired therapeutic or prophylactic activity,prior to use in humans. For example, in vitro assays which can be usedto determine whether administration of a specific ACE-2 bindingpolypeptide or composition of the present invention is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to, or otherwise administered, an ACE-2binding polypeptide or composition of the present invention, and theeffect of such an ACE-2 binding polypeptide or composition of thepresent invention upon the tissue sample is observed. In variousspecific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if an ACE-2 binding polypeptide or composition of the presentinvention has a desired effect upon such cell types. Preferably, theACE-2 binding polypeptides or compositions of the invention are alsotested in in vitro assays and animal model systems prior toadministration to humans.

[0519] ACE-2 binding polypeptides or compositions of the presentinvention for use in therapy can be tested for their toxicity insuitable animal model systems, including but not limited to rats, mice,chicken, cows, monkeys, and rabbits. For in vivo testing of an ACE-2binding polypeptide or composition's toxicity any animal model systemknown in the art may be used.

[0520] Efficacy in treating or preventing vasoconstriction may bedemonstrated by detecting the ability of an ACE-2 binding polypeptide orcomposition of the invention to prevent or treat hypertension, which canbe defined as a diastolic blood pressure greater than 90 mmHg and/or asystolic blood pressure of greater than 140 mmHg, as measured in anadult over 18 years of age. Since children and pregnant women have alower avaerage blood pressure, a mean diastolic blood pressure over 80mmHg and systolic blood pressure over 120 mmHg is considered indicativeof hypertension.

[0521] ACE-2 binding polypeptides or compositions of the invention canbe tested for the ability to inhibit or prevent smooth muscle cellproliferation, such as occurs in vascular stenosis and tumor formation,in in vitro, ex vivo, and in vivo assays. For example, cellularproliferation can be assayed by ³H-thymidine incorporation assays andtrypan blue cell counts.

[0522] ACE-2 binding polypeptides or compositions of the invention canbe tested for the ability to modulate inflammatory responses bycontacting cells involved in inflammatory responses, preferably humancells involved inflammatory responses (e.g., basophils and mast cells),with an ACE-2 binding polypeptide or composition of the invention tomodulate (i.e., increase or decrease) the inflammatory response. Theability of an ACE-2 binding polypeptide or composition of the inventionto modulate inflammatory responses can be assessed by detecting theproliferation of cells involved in the immune response (e.g., basophileand mast cells), detecting the activation of signalling molecules (e.g,bradykinin), detecting secretion of fluid and/or ions, detecting functiolaesa, detecting changes in calor or rubor at affected site, detectingthe expression of antigens, or detecting changes in blood flow to theaffected site. Techniques known to those of skill in the art can be usedfor measuring these activities. For example, antigen expression can beassayed, for example, by immunoassays including, but not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, immunohistochemistry radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays and FACS analysis. The activationof signaling molecules can be assayed, for example, by kinase assays andelectrophoretic shift assays (EMSAs). In a preferred embodiment, theability of an ACE-2 binding polypeptide or composition of the inventionto enhance or increase bradykinin concentrations and/or activity ismeasured.

[0523] ACE-2 binding polypeptides or compositions of the invention canalso be tested for their ability to alleviate of one or more symptomsassociated with cancer, a cardiovascular disorder (e.g., hypertension orhypotension), a neurological disorder, or a digestive disorder. Further,ACE-2 binding polypeptides or compositions of the invention can betested for their ability to increase the survival period of animalssuffering from disease or disorder, including cancer, a cardiovasculardisorder, a neurological disorder, or a digestive disorder. Techniquesknown to those of skill in the art can be used to analyze the functionof the ACE-2 binding polypeptides or compositions of the invention invivo.

[0524] Therapeutic/Prophylactic Compositions and Administration

[0525] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount ofACE-2 binding polypeptide (or fragment or variant thereof) orpharmaceutical composition of the invention, preferably an ACE-2 bindingpolypeptide of the invention. In a preferred aspect, an ACE-2 bindingpolypeptide or fragment or variant thereof is substantially purified(i.e., substantially free from substances that limit its effect orproduce undesired side-effects). The subject is preferably an animal,including but not limited to, animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably a human.

[0526] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0527] Various delivery systems are known and can be used to administerACE-2 binding polypeptide or fragment or variant thereof of theinvention, e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the ACE-2 bindingpolypeptide or ACE-2 binding polypeptide fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 262:4429-4432(1987)), construction of a nucleic acid as part of a retroviral or othervector, etc. Methods of introduction include, but are not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, and oral routes. The compositions may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

[0528] In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a protein, including an ACE-2 binding polypeptide, ofthe invention, care must be taken to use materials to which the proteindoes not absorb.

[0529] In another embodiment, the composition can be delivered in avesicle, in particular a liposome (see, Langer, Science, 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler, eds. (Liss, New York 1989), pp.353-365; Lopez-Berestein, ibid., pp. 317-327; see, generally, ibid.).

[0530] In yet another embodiment, the composition can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:201 (1987);Buchwald et al., Surgery, 88:507 (1980); Saudek et al., N. Engl. J.Med., 321:574 (1989)). In another embodiment, polymeric materials can beused (see, Medical Applications of Controlled Release, Langer and Wise,eds. (CRC Press, Boca Raton, Fla. 1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball,eds. (Wiley, New York 1984); Ranger and Peppas, Macromol. Sci. Rev.Macromol. Chem., 23:61 (1983); see also Levy et al., Science, 228:190(1985); During et al., Ann. Neurol., 25:35 1 (1989); Howard et al., J.Neurosurg., 7 1:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,e.g., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems arediscussed in the review by Langer (Science, 249:1527-1533 (1990)).

[0531] In a specific embodiment where the composition of the inventionis a nucleic acid encoding a protein, the nucleic acid can beadministered in vivo to promote expression of its encoded protein, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., by use of aretroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see, e.g.,Joliot et al., Proc. Natl. Acad. Sci. USA, 88:1864-1868 (1991)), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination.

[0532] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of anACE-2 binding polypeptide or a fragment thereof, and a pharmaceuticallyacceptable carrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations, andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,ed. (Mack Publishing Co., 1990). Such compositions will contain atherapeutically effective amount of the ACE-2 binding polypeptide orfragment thereof, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

[0533] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocamneto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0534] The compositions of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol,histidine, procaine, etc.

[0535] The amount of the composition of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0536] For ACE-2 binding polypeptides, the dosage administered to apatient is typically 0.1 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between 0.1mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kgto 10 mg/kg of the patient's body weight. Further, the dosage andfrequency of administration of therapeutic or pharmaceuticalcompositions of the invention may be reduced by enhancing uptake andtissue penetration (e.g., into the brain) of the ACE-2 bindingpolypeptides by modifications such as, for example, lipidation.

[0537] The ACE-2 binding polypeptides and ACE-2 binding polypeptidecompositions of the invention may be administered alone or incombination with other molecules including ACE-2. In further embodimentsof the invention, the ACE-2 binding polypeptides are administered incomplex with ACE-2. Preferably the ACE-2 binding polypeptide isradiolabelled or in complex with a radioisotope, toxin, or prodrug.Combinations may be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0538] The ACE-2 binding polypeptides and ACE-2 binding polypeptidecompositions of the invention may be administered alone or incombination with other adjuvants. Adjuvants that may be administeredwith the ACE-2 binding polypeptides and ACE-2 binding polypeptidecompositions of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, ACE-2 bindingpolypeptides and ACE-2 binding polypeptide compositions of the inventionare administered in combination with alum. In another specificembodiment, ACE-2 binding polypeptides and ACE-2 binding polypeptidecompositions of the invention are administered in combination withQS-21. Further adjuvants that may be administered with the ACE-2 bindingpolypeptides and ACE-2 binding polypeptide compositions of the inventioninclude, but are not limited to, Monophosphoryl lipid immunomodulator,AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, andVirosomal adjuvant technology. Vaccines that may be administered withthe ACE-2 binding polypeptides and ACE-2 binding polypeptidecompositions of the invention include, but are not limited to, vaccinesdirected toward protection against MMR (measles, mumps, rubella), polio,varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilusinfluenzae B, whooping cough, pneumonia, influenza, Lyme's Disease,rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis,rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23 TM.

[0539] The ACE-2 binding polypeptides and ACE-2 binding polypeptidecompositions of the invention may be administered alone or incombination with other therapeutic agents, including but not limited to,chemotherapeutic agents, antibiotics, antivirals, steroidal andnon-steroidal anti-inflammatories, conventional immunotherapeutic agentsand cytokines. Combinations may be administered either concomitantly,e.g., as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0540] In one embodiment, the ACE-2 binding polypeptides and ACE-2binding polypeptide compositions of the invention are administered incombination with antihypertensives including, but not limited to,diuretics, beta-blockers, calcium channel blockers, ACE inhibitors,angiotensin II receptor blockers, alpha blockers, combined alpha andbeta blockers, central agonists, peripheral adrenergic inhibitors, andblood vessel dilators.

[0541] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with diuretics including, but not limited to, bumetamide(Bumex®), chlorothiazide (Diuril®), chlorthalidone (Hygroton®),furosemide (Lasix®), hydrochlorothiazide (Esidrix®, Hydrodiuril®),mannitol, metolazone (Diulo®, Zaroxolyn®), amiloride andhydrochlorothiazide mix (Moduretic®), triamterene andhydrochlorothiazide mix (Dyazide®, Maxzide®), and potassium-sparringdiuretics, such as amiloride (Midamor®), spironolactone (Aldactone®),and triamterene (Dyrenium®).

[0542] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with beta blockers including, but not limited to,atenolol (Tenormin®), esmolol (Brevibloc®), labetalol (Normodyne®,Trandate®), metoprolol (Lopressor®), propranolol (Inderal®), sotalol(Betapace®), acebutolol (Sectral®), nadolol (Corgard®), pindolol(Visken®), and timolol (Blocadren®).

[0543] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with ACE inhibitors including, but not limited to,benazepril (Lotensin®), captopril (Capoten®), enalapril (Vasotec®),fosinopril (Monopril®), lisinopril (Zestril®, Prinivil®), and quinapril(Accupril®).

[0544] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with angiotensin II blockers including, but not limitedto losartan (Cozaar®).

[0545] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with calcium channel blockers including, but not limitedto, amlodipine (Norvasc®), diltiazem (Cardizem®), felodipine (Plendil®),isradipine (Dynacirc®), nicardipine (Cardene®), nifedipine (Procardia®),and verapamil (Calan®, Isoptin®).

[0546] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with alpha blockers including, but not limited to,doxazosin (Cardura®), prazosin (Minipress®), and tamsulosin (Flomax®).

[0547] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with central agonists including, but not limited to,clonidine (Catapres®), methyldopa (Aldomet®), guanabenz (Wytensin®), andguanfacine (Tenex®).

[0548] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with peripheral adrenergic inhibitors including, but notlimited to, reserpine, guanadrel (Hylorel®), and guanethidine(Ismelin®).

[0549] In a preferred embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredin combination with blood vessel dilators including, but not limited to,hydralzine (Apresoline®) and minoxidil (Loniten®).

[0550] In another preferred embodiment, the ACE-2 binding polypeptidesand ACE-2 binding polypeptide compositions of the invention areadministered in combination with vasodilating agents including, but notlimited to, alprostadil (PGE-1, prostaglandin E-1, Prostin VRPediatric®), amyl nitrite, dipyridamole (Persantin®), epoprostenol(Flolan®), isosorbide dinitrate (Isordil®, Sorbitrate®), isosorbidemonomitrate (IMDUR®), nimodipine (nimotop®), nitric oxide gas (INOmax®),nitroglycerin (glyceryl trinitrate, Nitro-Dur®, Nitroligual®,Nitrostat®, NTG, Transderm-Nirto®), papaverine, and tolazoline(Priscoline®).

[0551] Antilipemic agents that may be administered in combination withthe ACE-2 binding polypeptides and ACE-2 binding polypeptidecompositions of the present invention include, but are not limited to,atorvastatin (Lipitor®), cholestyramine (Questran®), colestipol(Colestid®), fluvastatin (Lescol®), gemfibrozil (Lopid®), niacin(nicotinic acid), prevastatin (Pravachol®), and simvastatin (Zocor®).

[0552] In another preferred embodiment, a therapeutically effectiveamount of angiotensin 1-9 is administered in combination with atherapeutically effective amount of angiotensin II or other hypotensiveagents (see Example 9).

[0553] Additionally, in another embodiment, the ACE-2 bindingpolypeptides and ACE-2 binding polypeptide compositions of the inventionare administered in combination with intravascular radiation to prevent,treat, or ameliorate restenosis. In a preferred embodiment, the ACE-2binding polypeptides and ACE-2 binding polypeptide compositions of theinvention are administered in combination with beta-emittingphosphorus-32. In another preferred ambodiment, the ACE-2 bindingpolypeptides and ACE-2 binding polypeptide compositions of the inventionare administered in combination with iridium-192.

[0554] In a further embodiment, the ACE-2 binding polypeptides and ACE-2binding polypeptide compositions of the invention are administered incombination with analgesics and anti-inflammatory agents. Analgesicagents that may be administered in combination with the ACE-2 bindingpolypeptides and ACE-2 binding polypeptide compositions of the inventioninclude, but are not limited to, opioids, such as codeine, fentanyl(Actig®, Duragesic®, Oralet®, Sublimaze®), hydromorphone (Dilaudid®),meperidine (Demerol®), methadone (Dolophine®), morphine (Duramorph®,Infomorph®, morphine sulfate, MSO4), oxycodone, remifentanil (Ultiva®),and sufentanil (Sufenta®); opiate partial agonists, such as butorphanol(Stadol®), nalbuphine (Nubain®), and tramadol (Ultram®).Anti-inflammatory agents that may be administered in combination withthe ACE-2 binding polypeptides and ACE-2 binding peptide compositions ofthe invention include, but are not limited to, NSAIDs (non-steroidalanti-inflammatory drugs), such as aspirin (ASA®. Empirin®), cholinemagnesium trisalicylate (Trilisate®), diclofenac, diflunisal,fenoprofen, flurbiprofin (Ocufen®), ibuprofin (Advil®, Motrin®, Rufen®),indomethacin (Indocin®), ketoprofen, meclofenamate, nabumetone(Relafen®), naproxen (Naprosyn®), naproxen sodium (Aleve®, Anaprox®),oxaprozin, phenylbutazone, piroxicam (Feldene®), salsalate (Disalcid®),sulindac (Clinoril®), tolmetin, celecoxib (Celebrex®), and ketorolac(Toradol®).

[0555] In an even further embodiment, the ACE-2 binding polypeptides andACE-2 binding polypeptide compositions of the invention are administeredalone or in combination with other anti-inflammatory agents.Anti-inflammatory agents that may be administered with the ACE-2 bindingpolypeptides and ACE-2 binding polypeptide compositions of the inventioninclude, but are not limited to, glucocorticoids and the nonsteroidalanti-inflammatories, aminoarylcarboxylic acid derivatives, arylaceticacid derivatives, arylbutyric acid derivatives, arylcarboxylic acids,arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acidderivatives, thiazinecarboxamides, e-acetamidocaproic acid,S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine,bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone,guaiazulene, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,pifoxime, proquazone, proxazole, and tenidap.

[0556] In another embodiment, compostions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the ACE-2 bindingpolypeptides and ACE-2 binding polypeptide compositions of the inventioninclude, but are not limited to, antibiotic derivatives (e.g.,doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens(e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU,methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0557] In a specific embodiment, ACE-2 binding polypeptides and ACE-2binding polypeptide compositions of the invention are administered incombination with CHOP (cyclophosphamide, doxorubicin, vincristine, andprednisone) or any combination of the components of CHOP. In oneembodiment, the compositions of the invention are administered incombination with anti-CD20 antibodies, human monoclonal anti-CD20antibodies. In another embodiment, the compositions of the invention areadministered in combination with anti-CD20 antibodies and CHOP, oranti-CD20 antibodies and any combination of one or more of thecomponents of CHOP, particularly cyclophosphamide and/or prednisone. Ina specific embodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituximab and CHOP, or Rituximab and anycombination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. In a specific embodiment,compositions of the invention are administered in combination withtositumomab. In a further embodiment, compositions of the invention areadministered with tositumomab and CHOP, or tositumomab and anycombination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. The anti-CD20 antibodies mayoptionally be associated with radioisotopes, toxins or cytotoxicprodrugs.

[0558] In another specific embodiment, the compositions of the inventionare administered in combination Zevalin™. In a further embodiment,compositions of the invention are administered with Zevalin™ and CHOP,or Zevalin™ and any combination of one or more of the components ofCHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may beassociated with one or more radisotopes. Particularly preferred isotopesare ⁹⁰Y and ¹¹¹In.

[0559] Additionally, the ACE-2 binding polypeptides and ACE-2 bindingpolypeptide compositions of the invention may be administered alone orin combination with other therapeutic regimens, including but notlimited to, radiation therapy. Such combinatorial therapy may beadministered sequentially and/or concomitantly.

[0560] Kits for Detecting and/or Quantitating ACE-2 or ACE-2-LikePolypeptides

[0561] The present invention is also directed to an assay kit which canbe useful in screening for the presence of ACE-2 and/or quantitatingACE-2 concentrations in a fluid, such as, for example, a biologicalfluid (e.g., blood, serum, synovial fluid).

[0562] In a particular embodiment of the present invention, an assay kitis contemplated which comprises in one or more containers one or moreACE-2 binding polypeptides of the invention and optionally, a detectionmeans for determining the presence of an ACE-2-ACE-2 binding polypeptideinteraction or the absence thereof. The kit further optionally containsACE-2 protein that may be used, for example as a control. The ACE-2binding polypeptide may be free or expressed on the surface of a phage.

[0563] In a specific embodiment, either the ACE-2 binding polypeptide orthe ACE-2 protein is labeled. As further discussed herein, a wide rangeof labels can be used accordance with the present invention, includingbut not limited to conjugating the recognition unit to biotin byconventional means. Alternatively, the label may comprise, e.g., afluorogen, an enzyme, an epitope, a chromogen, or a radionuclide.Preferably, the biotin is conjugated by covalent attachment to eitherthe ACE-2 binding polypeptide or the ACE-2 protein. Preferably, theACE-2 binding polypeptide is immobilized on a solid support. Thedetection means employed to detect the label will depend on the natureof the label and can be any known in the art, e.g., film to detect aradionuclide, an enzyme substrate that gives rise to a detectable signalto detect the presence of an enzyme, antibody to detect the presence ofan epitope, etc.

[0564] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In one preferred embodiment the kit comprises a vialcontaining ACE-2 binding polypeptides conjugated to a toxin or a label(as described herein). Such conjugated binding polypeptide may be usedto kill a particular population of cells or to quantitate a particularpopulation of cells. In a preferred embodiment, such conjugated ACE-2binding polypeptides are used to kill cells expressing themembrane-bound form of ACE-2. In another preferred embodiment, suchconjugated ACE-2 binding polypeptides are used to quantitate cellsexpressing the membrane-bound form of ACE-2.

[0565] In one embodiment, the present invention provides apharmaceutical pack or kit comprising a first container containing ACE-2binding polypetides attached to a macrocyclic chelator (e.g., DOTA), asecond container containing ACE-2 polypeptide, and a third containercontaining radiometal ions (e.g., ⁹⁰Y, ¹¹¹In, or ¹³¹I,). The contents ofthese containers could then be mixed, and incubated for a period of timeto allow the reagents to associate with one another, prior toadministration to a patient. In addition it may also be desirable topurify the ACE-2/ACE-2 binding polypeptide-macrocyclicchelator/radiometal ion complexes from excess reagents that did not forminto complexes, for example, by filtration in a spin column based on asize exclusion principle (e.g. Bio-Spin columns available from BioradLaboratories, Inc.) Thus in another embodiment, the present inventionprovides a pharmaceutical pack or kit comprising a first containercontaining ACE-2 binding polypetides attached to a macrocyclic chelator(e.g., DOTA), a second container containing ACE-2 polypeptide, and athird container containing radiometal ions (e.g., ⁹⁰Y, ¹¹¹In, or ¹³¹I),and a means for purifying ACE-2/ACE-2 binding polypeptide-macrocyclicchelator/radiometal ion complexes.

[0566] In other embodiments, the present invention provides apharmaceutical pack or kit comprising a first container containing ACE-2and ACE-2 binding poypeptides, and a second container containingradiometal ions (e.g., ⁹⁰Y, ¹¹¹In, or ¹³¹I). In a specific embodiment,the present invention provides a pharmaceutical pack or kit comprising afirst container containing ACE-2 and ACE-2 binding poypeptides, a secondcontainer containing radiometal ions (e.g., ⁹⁰Y, ¹¹¹In, or ¹³¹I), and ameans for purifying ACE-2/ACE-2 binding polypeptide-macrocyclicchelator/radiometal ion complexes.

[0567] In still other embodiments, the present invention provides apharmaceutical pack or kit comprising a first container containing ACE-2binding polypetides attached to a macrocyclic chelator (e.g., DOTA) anda second container containing radiometal ions (e.g., ⁹⁰Y, ¹¹¹In, or¹³¹I,). In a specific embodiment, the present invention provides apharmaceutical pack or kit comprising a first container ACE-2 bindingpolypetides attached to a macrocyclic chelator (e.g., DOTA), a secondcontainer containing radiometal ions (e.g., ⁹⁰Y, ¹¹¹In, or ¹³¹I,) and ameans for purifying ACE-2 binding polypeptide-macrocyclicchelator/radiometal ion complexes.

[0568] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an ACE-2 binding polypeptideof the invention, preferably a purified ACE-2 binding polypeptide, inone or more containers. In an alterative embodiment, a kit comprises anACE-2 binding polypeptide fragment that specifically binds to ACE-2. Ina specific embodiment, the kits of the present invention contain asubstantially isolated ACE-2 polypeptide as a control. Preferably, thekits of the present invention further comprise a control bindingpolypeptide which does not react with ACE-2. In another specificembodiment, the kits of the present invention contain a means fordetecting the binding of an ACE-2 binding polypeptide to ACE-2 (e.g.,the ACE-2 binding polypeptide may be conjugated to a detectablesubstrate such as a fluorescent compound, an enzymatic substrate, aradioactive compound or a luminescent compound, or a second antibodywhich recognizes the ACE-2 binding polypeptide may be conjugated to adetectable substrate). In specific embodiments, the kit may include arecombinantly produced or chemically synthesized ACE-2. The ACE-2provided in the kit may also be attached to a solid support. In a morespecific embodiment the detecting means of the above-described kitincludes a solid support to which ACE-2 is attached. Such a kit may alsoinclude a non-attached reporter-labeled anti-ACE-2 binding polypeptideantibody. In this embodiment, binding of the ACE-2 binding polypeptideto ACE-2 can be detected by binding of the said reporter-labeledantibody. Alternatively, or in addition, the detecting means may includea labeled, competing antigen.

[0569] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing ACE-2 or ACE-2-likepolypeptides. The diagnostic kit includes a substantially isolated ACE-2binding polypeptide specifically reactive with ACE-2 target, and meansfor detecting the binding of ACE-2 target to the ACE-2 bindingpolypeptide. In one embodiment, the ACE-2 binding polypeptide isattached to a solid support.

[0570] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound ACE-2 binding polypeptideaccording to the present invention. After ACE-2 binds to a specificACE-2 binding polypeptide, the unbound serum components are removed bywashing, reporter-labeled anti-ACE-2 binding polypeptide antibody isadded, unbound anti-ACE-2 binding polypeptide antibody is removed bywashing, and a reagent is reacted with reporter-labeled anti-ACE-2binding polypeptide antibody to bind reporter to the reagent inproportion to the amount of bound ACE-2 binding polypeptide on the solidsupport. Typically, the reporter is an enzyme which is detected byincubating the solid phase in the presence of a suitable fluorometric,luminescent or colorimetric substrate.

[0571] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated ACE-2 binding polypeptides.

[0572] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant ACE-2, and a reporter-labeled anti-ACE-2binding polypeptide antibody for detecting surface-bound anti-ACE-2binding polypeptide.

[0573] Methods of Screening for ACE-2 Binding Molecules

[0574] The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind ACE-2, and the ACE-2 bindingmolecules identified thereby. This method comprises the steps of:

[0575] (a) contacting ACE-2 or ACE-2-like polypeptide with a pluralityof molecules; and

[0576] (b) identifying molecule(s) that binds the ACE-2 or ACE-2-likepolypeptide.

[0577] The step of contacting the ACE-2 protein or ACE-2-like proteinwith the plurality of molecules may be effected in a number of ways. Forexample, one may contemplate immobilizing ACE-2 target on a solidsupport and bringing a solution of the plurality of molecules in contactwith the immobilized ACE-2 target. Such a procedure would be akin to anaffinity chromatographic process, with the affinity matrix beingcomprised of the immobilized ACE-2 protein or ACE-2-like polypeptide.The molecules having a selective affinity for the ACE-2 or ACE-2-likepolypeptide can then be purified by affinity selection. The nature ofthe solid support, process for attachment of the ACE-2 or ACE-2-likepolypeptide to the solid support, solvent, and conditions of theaffinity isolation or selection are largely conventional and well knownto those of ordinary skill in the art.

[0578] Alternatively, one may also separate a plurality of polypeptidesinto substantially separate fractions comprising a subset of orindividual polypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” using an ACE-2 target protein, optionally in the presence ofan inducer should one be required for expression, to determine if anyselective affinity interaction takes place between the ACE-2 targetprotein and the individual clone. Prior to contacting the ACE-2 targetprotein with each fraction comprising individual polypeptides, thepolypeptides could first be transferred to a solid support foradditional convenience. Such a solid support may simply be a piece offilter membrane, such as one made of nitrocellulose or nylon. In thismanner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity for ACE-2or ACE-2-like polypeptide. Furthermore, the amino acid sequence of thepolypeptide having a selective affinity for the ACE-2 protein orACE-2-like protein can be determined directly by conventional means, orthe coding sequence of the DNA encoding the polypeptide can frequentlybe determined more conveniently. The primary amino acid sequence canthen be deduced from the corresponding DNA sequence. If the amino acidsequence is to be determined from the polypeptide itself, one may usemicrosequencing techniques. The sequencing technique may include massspectroscopy.

[0579] In certain situations, it may be desirable to wash away any ACE-2or ACE-2-like polypeptide, or alterntatively, unbound polypeptides, froma mixture of ACE-2 or ACE-2-like polypeptide and the plurality ofpolypeptides prior to attempting to determine or to detect the presenceof a selective affinity interaction. One or more such a wash steps maybe particularly desirable when the ACE-2 or ACE-2-like polypeptide orthe plurality of polypeptides is bound to a solid support.

[0580] The plurality of molecules provided according to this method maybe provided by way of diversity libraries, such as random orcombinatorial peptide or non-peptide libraries which can be screened formolecules that specifically bind to ACE-2. Peptide libraries may bedesigned such that the polypeptides encoded by the libraries areautomatically fused to a polypeptide linker moiety, for example. Manylibraries are known in the art that can be used, e.g., chemicallysynthesized libraries, recombinant (e.g., phage display libraries), andin vitro translation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., Science, 251:767-773 (1991);Houghten et al., Nature, 354:84-86 (1991); Lam et al., Nature, 354:82-84(1991); Medynski, Bio/Technology, 12:709-710 (1994); Gallop et al., J.Medicinal Chemistry, 37(9):1233-1251 (1994); Ohlmeyer et al., Proc.Natl. Acad. Sci. USA, 90:10922-10926 (1993); Erb et al., Proc. Natl.Acad. Sci. USA, 91:11422-11426 (1994); Houghten et al., Biotechniques,13:412 (1992); Jayawickreme et al., Proc. Natl. Acad. Sci. USA,91:1614-1618 (1994); Salmon et al., Proc. Natl. Acad. Sci. USA,90:11708-11712 (1993); PCT publication WO 93/20242; and Brenner andLerner, Proc. Natl. Acad. Sci. USA, 89:5381-5383 (1992).

[0581] Examples of phage display libraries are described in Scott andSmith, Science, 249:386-390 (1990); Devlin et al., Science, 249:404-406(1990); Christian et al., J. Mol. Biol., 227:711-718 (1992); Lenstra, J.Immunol. Meth., 152:149-157 (1992); Kay et al., Gene, 128:59-65 (1993);and PCT publication WO 94/18318.

[0582] In vitro translation-based libraries include but are not limitedto those described in PCT publication WO 91/05058 and Mattheakis et al.,Proc. Natl. Acad. Sci. USA, 91:9022-9026 (1994).

[0583] By way of examples of non-peptide libraries, a benzodiazepinelibrary (see, e.g., Bunin et al., Proc. Natl. Acad. Sci. USA,91:4708-4712 (1994) can be adapted for use. Peptoid libraries (Simon etal., Proc. Natl. Acad. Sci. USA, 89:9367-9371 (1992)) can also be used.Another example of a library that can be used, in which the amidefunctionalities in peptides have been permethylated to generate achemically transformed combinatorial library, is described by Ostresh etal. (Proc. Natl. Acad. Sci. USA, 91:11138-11142 (1994)).

[0584] The variety of non-peptide libraries that are useful in thepresent invention is great. For example, Ecker and Crooke,Bio/Technology, 13:351-360 (1995) list benzodiazepines, hydantoins,piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones,arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines,aminimides, and oxazolones as among the chemical species that form thebasis of various libraries.

[0585] Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

[0586] Non-peptide oligomer libraries utilize a large number of monomersthat are assembled together in ways that create new shapes that dependon the order of the monomers. Among the monomer units that have beenused are carbamates, pyrrolinones, and morpholinos. Peptoids,peptide-like oligomers in which the side chain is attached to the alphaamino group rather than the alpha carbon, form the basis of anotherversion of non-peptide oligomer libraries. The first non-peptideoligomer libraries utilized a single type of monomer and thus containeda repeating backbone. Recent libraries have utilized more than onemonomer, giving the libraries added flexibility.

[0587] Screening the libraries can be accomplished by any of a varietyof commonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, Adv. Exp.Med. Biol., 251:215-218 (1989); Scott and Smith, Science, 249:386-390(1990); Fowlkes et al., BioTechniques, 13:422-427 (1992); Oldenburg etal., Proc. Natl. Acad. Sci. USA, 89:5393-5397 (1992); Yu et al., Cell,76:933-945 (1994); Staudt et al., Science, 241:577-580 (1988); Bock etal., Nature, 355:564-566 (1992); Tuerk et al., Proc. Natl. Acad. Sci.USA, 89:6988-6992 (1992); Ellington et al., Nature, 355:850-852 (1992);U.S. Pat. Nos. 5,096,815; 5,223,409; and 5,198,346, all to Ladner etal.; Rebar and Pabo, Science, 263:671-673 (1993); and PCT publication WO94/18318.

[0588] In a specific embodiment, screening to identify a molecule thatbinds ACE-2 can be carried out by contacting the library members withACE-2 or ACE-2-like polypeptide immobilized on a solid phase andharvesting those library members that bind to the ACE-2 or ACE-2-likepolypeptide. Examples of such screening methods, termed “panning”techniques are described by way of example in Parmley and Smith, Gene,73:305-318 (1998); Fowlkes et al., BioTechniques, 13:422-427 (1992); PCTpublication WO 94/18318; and in references cited therein.

[0589] In another embodiment, the two-hybrid system for selectinginteracting proteins in yeast (Fields and Song, Nature, 340:245-246(1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991))can be used to identify molecules that specifically bind to ACE-2 orACE-2-like polypeptides.

[0590] Where the ACE-2 binding molecule is a polypeptide, thepolypeptide can be conveniently selected from any peptide library,including random peptide libraries, combinatorial peptide libraries, orbiased peptide libraries. The term “biased” is used herein to mean thatthe method of generating the library is manipulated so as to restrictone or more parameters that govern the diversity of the resultingcollection of molecules, in this case peptides.

[0591] Thus, a truly random peptide library would generate a collectionof peptides in which the probability of finding a particular amino acidat a given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid, that certain aminoacid positions in a peptide remain fixed (e.g., as cysteine), or thatpositions 4, 8, and 9, for example, of a decapeptide library be limitedto permit several but less than all of the twenty naturally-occurringamino acids. Clearly, many types of biases can be contemplated, and thepresent invention is not restricted to any particular bias. Furthermore,the present invention contemplates specific types of peptide libraries,such as phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

[0592] As mentioned above, in the case of an ACE-2 binding molecule thatis a polypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, an ACE-2 binding polypeptide has in the range of 15-100amino acids, or 20-50 amino acids.

[0593] The selected ACE-2 binding polypeptide can be obtained bychemical synthesis or recombinant expression.

[0594] The specific ACE-2 binding polypeptides disclosed herein wereisolated using phage display technology, to identify ACE-2 bindingpolypeptides exhibiting particular preselected binding properties. TheseACE-2 binding polypeptides were isolated initially by screening ninephage display libraries, that is, populations of recombinantbacteriophage transformed to express an exogenous recombinantpolypeptide on their surface. In order to isolate new polypeptidebinding moieties for a particular target, such as ACE-2, screening ofpeptide libraries, for example using phage display techniques, isespecially advantageous, in that very large numbers (e.g., 5×10⁹) ofpotential binders can be tested and successful binders isolated in ashort period of time.

[0595] In order to prepare a phage library of potential bindingpolypeptides to screen for members of the library that are ACE-2 bindingpolypeptides, a candidate binding domain is selected to serve as astructural template for the polypeptides to be displayed in the library.The phage library is made up of polypeptide analogues of this templateor “parental binding domain.” The parental binding domain is apolypeptide molecule that may be a naturally occurring or syntheticprotein or polypeptide, or polypeptide region or domain of a protein.The parental binding domain may be selected based on knowledge of aknown interaction between the parental binding domain and a targetprotein, but this is not critical. In fact, it is not essential that theparental binding domain have any affinity for a target at all becauseits purpose is to provide a structure from which a multiplicity ofpolypeptide analogues (a “library”) can be generated, which multiplicityof polypeptide analogues will include one or more binding polypeptidesthat exhibit the desired binding and release properties with respect toACE-2 target proteins (and any other properties selected).

[0596] Knowledge of the exact polypeptide that will serve as theparental binding domain, or knowledge of a class of proteins or domainsto which the parental binding domain belongs can be useful indetermining the conditions under which ACE-2 binding polypeptidesoptimally bind ACE-2 target proteins as well as the conditions underwhich ACE-2 binding polypeptides optimally release ACE-2 targetproteins. Similarly, the binding and/or release conditions may beselected with regard to known interactions between a binding domain andthe ACE-2 target protein, for example, to favor the interaction underthe binding and/or release conditions, or they may be selected withoutregard to such known interactions. Likewise, the parental binding domaincan be selected taking into account a desired binding and/or releasecondition or not. It is understood that if the binding domain analoguesof a library are unstable under a proposed or desired binding or releasecondition, no useful binding polypeptides may be obtained.

[0597] In selecting the parental binding domain, the most importantconsideration is how the analogue domains will be presented to the ACE-2target protein, that is, in what conformations the ACE-2 target and thepolypeptide analogues will contact one another. In preferredembodiments, for example, the polypeptide analogues will be generated byinsertion of synthetic DNA encoding the polypeptide analogue into areplicable genetic package, resulting in display of the domain on thesurface of a microorganism, such as M13 phage, using techniques asdescribed in Kay et al., Phage Display of Peptides and Proteins: ALaboratory Manual (Academic Press, Inc.; San Diego 1996) and U.S. Pat.No. 5,223,409 (Ladner et al.), incorporated herein by reference. Forformation of phage display libraries, it is preferred to use structuredpolypeptides as the parental binding domain or template, as opposed tounstructured, linear peptides. Mutation of surface residues in a proteindomain or polypeptide molecule will usually have little effect on theoverall structure or general properties (such as size, stability, andtemperature of denaturation) of the protein; while at the same timemutation of surface residues may profoundly affect the bindingproperties of the molecule. The more tightly a polypeptide segment isconstrained, the less likely it is to bind to any particular target. Ifit does bind, however, the binding is likely to be tighter and morespecific. Thus, it is preferred to select a parental binding domainwherein the parental polypetide has structure and, thereby in turn,select a structure for the polypeptide analogues of the library, whichis constrained within a framework having some degree of rigidity.

[0598] Preferably the protein domain that is used as the template orparental domain for generating the library of domain analogues will be apeptide molecule that is a relatively small protein or polypeptide.Small polypeptides offer several advantages over large proteins: First,the mass per binding site is reduced. Highly stable protein domainshaving low molecular weights, for example, Kunitz domains (˜7kilodaltons, kDa), Kazal domains (˜7 kDa), Cucurbida maxima trypsininhibitor (CMTI) domains (˜3.5 kDa), and endothelin (˜2 kDa), can showmuch higher binding per gram than do antibodies (150 kDa) or singlechain scFv antibodies (30 kDa). Second, the possibility of non-specificbinding is reduced because there is less molecular surface available fornonspecific binding. Third, small polypeptides can be engineered to haveunique tethering sites in a way that is impracticable for largerproteins or antibodies. For example, small proteins and polypeptides canbe engineered to have lysines only at sites suitable for tethering to achromatography matrix. This is not feasible for antibodies. Fourth, aconstrained polypeptide structure is more likely to retain itsfunctionality when transferred (with the structural domain intact) fromone framework to another. For instance, the binding domain structure islikely to be transferable from the framework used for presentation in alibrary, such as displayed on a phage, to an isolated protein removedfrom the presentation framework or immobilized on a chromatographicsubstrate.

[0599] In specific embodiments, the ACE-2 binding polypeptides of theinvention are immobilized. ACE-2 binding polypeptide molecules accordingto the invention may be immobilized, for example, on chromatographicsupport materials to form efficient ACE-2 separation or affinitychromatographic media. Immobilized ACE-2 binding polypeptides of theinvention have uses that include, but are not limted to, detecting,isolating or removing ACE-2 target proteins from solutions. One strategyfor generating ACE-2 binding polypeptide molecules that can beimmobilized, for example, on matrices, resins, or supports, involvesselecting appropriate binding domain templates such that ACE-2 bindingpolypeptide molecules are generated that have one or more amino acidsthat may be used to covalently link the ACE-2 binding polypeptide to achromatographic resin or substrate to form an affinity resin. Similarly,the N-terminal amino group or the C-terminal carboxyl group of a peptidemolecule may be modified by adding a capping group to render it inert ora functional group, which permits linkage to a support medium. Forexample, the C-terminal carboxyl group of a protein domain may beconverted to an amide or a hydrazide (—NH—NH₂) group for reaction withan aldehyde-functional substrate or other amine-reactive substrate. Thistechnique is preferred. Another preferred modification of ACE-2 bindingpolypeptides useful for linking an ACE-2 binding polypeptide molecule ofthe invention to a chromatography material is a polypeptide linkercomprising, or alternatively consisting of, the amino acid sequencePro-Gly-Pro-Glu-Gly-Gly-Gly-Lys (SEQ ID NO:13).

[0600] In one non-limiting example of a screening procedure to obtainACE-2 binding polypeptides encompassed by the invention, the phage in aphage display library are contacted with and allowed to bind an ACE-2target protein that is immobilized on a solid support. Those phage thatdisplay non-binding polypeptides are separated from those that bind theACE-2 target protein. Any of various techniques known in the art may beapplied to dissociate the bound phage from the immobilized ACE-2protein, and to collect and/or amplify the phage and/or their nucleicacid contents. Using these techniques it is possible to identify a ACE-2binding phage that is about 1 in 20 million in the population.Libraries, displaying 10-20 million or more potential binding peptidemolecules each, are rapidly screened to find high-affinity ACE-2 bindingpolypeptides.

[0601] In each round of screening, the diversity of a population fallsuntil only efficient binding polypeptides remain, that is, the processconverges. Typically, a phage display library will contain severalclosely related binding polypeptides (10 to 50 different bindingpolypeptides out of 10 million). Indications of convergence includeincreased binding (measured by phage titers) and recovery of closelyrelated sequences. After a first set of binding polypeptide molecules isidentified, the sequence information can be used to design otherlibraries biased for members having additional desired properties, forexample, discrimination between different forms of ACE-2 (e.g., themembrane form and the soluble form of ACE-2) and fragments thereof, ordiscrimination between ACE-2 and closely related impurities in a feedstream.

[0602] Such techniques make it possible not only to screen a largenumber of potential binding polypeptides, but make it practical torepeat the binding and elution cycles and to build secondary, biasedlibraries for screening polypeptide analogue-displaying phage that meetspecific criteria. Using these techniques, a polypeptide analogue biasedlibrary may be screened to reveal members that bind tightly, that is,have high affinity for ACE-2 target protein, under the screeningconditions.

[0603] In the present invention target ACE-2 protein molecules werebiotinylated and then bound to streptavidin-coated magnetic particles.Eight phage display libraries of different design were screened for theability to bind the immobilized ACE-2. Six of the libraries werecharacterized by M13 phage displaying a variegated exogenous peptideloop of different lengths and overall structure: The TN6/6 library wasconstructed to display a single microprotein binding loop contained in a12-amino acid template. The TN7/4 library was constructed to display asingle microprotein binding loop contained in a 13-amino acid template.The TN8/9 library was constructed to display a single microproteinbinding loop contained in a 14-amino acid template. The TN9/4 librarywas constructed to display a single microprotein binding loop containedin a 15-amino acid template. The TN10/9 library was constructed todisplay a single microprotein binding loop contained in a 16-amino acidtemplate. The TN12/1 library was constructed to display a singlemicroprotein binding loop contained in an 18-amino acid template. Twocommercially available linear phage display libraries were alsoscreened, designated PhD 7 and PhD 12 (New England Biolabs). The PhD 7library displayed a linear random-sequence 7-mer and the PhD 12 librarydisplayed a random-sequence 12-mer

[0604] ACE-2 binding phage were not isolated from either of thecomercially available libraries, PhD 7 and PhD 12.

[0605] After analysis of the sequences isolated from the libraryscreenings, several families of ACE-2 binding peptides were defined. Theamino acid sequences of the ACE-2-binding “hits” are set forth inExample 1 (infra).

[0606] As it within the scope of the present invention to screen phagelibraries that bind one or more of the various forms of ACE-2, thefollowing outlines some assays that may be used in screening for ACE-2binding polypeptides that bind the soluble form of ACE-2, themembrane-bound form of ACE-2, or both the soluble and the membrane-boundforms of ACE-2. Assays to determine the specificity of bindingpolypeptides for different forms of a protein are commonly known in theart and may be readily adapted for determining the specificity of ACE-2binding polypeptides for different forms of ACE-2.

[0607] ACE-2 binding polypeptides of the invention (including moleculescomprising, or alternatively consisting of, ACE-2 binding polypeptidefragments or variants thereof) may be screened in a variety of assays toidentify those ACE-2 binding polypeptides that specifically bind to thesoluble form of ACE-2. ACE-2 binding polypeptides may be assayed inneutralization assays described herein (see Example 4) or otherwiseknown in the art. For example, ACE-2 binding polypeptides may be testedfor their ability to inhibit soluble ACE-2 from binding an ACE-2substrate.

[0608] ACE-2 binding polypeptides of the invention (including moleculescomprising, or alternatively consisting of, ACE-2 binding polypeptidefragments or variants thereof) may be screened in a variety of assayscommonly known in the art to identify those ACE-2 binding polypeptidesthat specifically bind to the membrane-bound form of ACE-2. For example,ACE-2 binding polypeptides may be assayed for binding ACE-2 proteinpresent on cell membranes of cells that express ACE-2.

[0609] ACE-2 binding polypeptides of the invention (including moleculescomprising, or alternatively consisting of, ACE-2 binding polypeptidefragments or variants) may be screened in a variety of assays toidentify those ACE-2 binding polypeptides or ACE-2 binding polypeptidefragments or variants that specifically bind to the soluble form andmembrane-bound form of ACE-1. This can readily be determined byperforming assays to distinguish binding to the soluble form and assaysto distinguish binding to the membrane-bound form (such as the assaysdescribed herein or otherwise known in the art), and identifying theACE-2 binding polypeptides that bind both forms.

[0610] Additionally, ACE-2 binding polypeptides of the invention may bescreened for the ability to inhibit, stimulate or not significantlyalter ACE-2 activity, e.g., the ability of ACE-2 to bind to itssubstrate (e.g., angiotensin).

[0611] Anti-ACE-2 Binding Polypeptide Antibodies

[0612] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind an ACE-2binding polypeptide of the present invention (as determined byimmunoassays well known in the art for assaying specificantibody-antigen binding). Antibodies of the invention include, but arenot limited to, polyclonal, monoclonal, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-id) antibodies (including, e.g., anti-id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. Immunoglobulins may have both a heavy and lightchain. In specific embodiments, the immunoglobulin molecules of theinvention are IgG1. In other specific embodiments, the immunoglobulinmolecules of the invention are IgG4. An array of IgG, IgE, IgM, IgD,IgA, and IgY heavy chains may be paired with a light chain of the kappaor lambda forms.

[0613] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 to Kucherlapati et al.

[0614] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715, WO 92/08802, WO 91/00360, WO 92/05793; Tutt etal., J. Immunol., 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.,148:1547-1553 (1992).

[0615] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of an ACE-2 binding polypeptideof the present invention which they recognize or specifically bind.Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind ACE-2 binding polypeptides ofthe present invention, and allows for the exclusion of the same.

[0616] In further preferred, nonexclusive embodiments, the antibodies ofthe invention (e.g., anti-idiotypic antibodies) inhibit one or morebiological activities of ACE-2 through specific binding to ACE-2. Inanother preferred embodiment, the antibody of the invention inhibitsACE-2-mediated vasoconstriction.

[0617] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other ACE-2 binding polypeptide of the present invention areincluded. Antibodies that bind polypeptides with at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, at least 60%, at least 55%, and at least 50% identity (ascalculated using methods known in the art and described herein) to anACE-2 binding polypeptide of the present invention are also included inthe present invention. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to an ACE-2 binding polypeptide of the presentinvention are also included in the present invention. Further includedin the present invention are antibodies which bind polypeptides encodedby polynucleotides, the complement of which hybridize to apolynucleotides of the present invention under stringent hybridizationconditions (as described herein). Antibodies of the present inventionmay also be described or specified in terms of their binding affinity toan ACE-2 binding polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10 ⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M,5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

[0618] The invention also provides antibodies that competitively inhibitbinding of an antibody to an ACE-2 binding polypeptide of the inventionas determined by any method known in the art for determining competitivebinding. In preferred embodiments, the antibody competitively inhibitsbinding to the ACE-2 binding polypeptide by at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, orat least 50%.

[0619] Antibodies of the present invention (e.g., anti-idiotypicantibodies) may act as agonists or antagonists of ACE-2 or alternativelymay not significantly alter ACE-2 mediated activity. For example, thepresent invention includes antibodies (e.g., anti-idiotypic antibodies)which disrupt ACE-2/ACE-2 substrate (e.g., angiotensin, bradykinin,tachykinin, endothelin, neurotensin, or Substance P) interactions eitherpartially or fully. In another example, antibodies of the inventionenhance ACE-2/ACE-2 receptor interactions either partially or fully.Such activity may be the result of, for example, the antibody binding toan ACE-2 binding polypeptide of the invention, or alternatively as aresult of direct binding of the antibody (e.g., an anti-idiotypicantibody to ACE-2).

[0620] Preferrably, antibodies of the present invention bind an ACE-2binding polypeptide disclosed herein, a portion thereof, or an antibodythat binds an ACE-2 binding polypeptide disclosed herein, or a portionthereof. The invention features both ACE-2 binding polypeptide-specificantibodies and antibodies that are specific to ACE-2 bindingpolypeptide/ACE-2 complexes. The invention features antibodies thatenhance ACE-2/ACE-2 binding polypeptide binding and/or ACE-2/ACE-2substrate binding. The invention also features antibodies that do notinhibit or reduce ACE-2/ACE-2 binding polypeptide binding and/orACE-2/ACE-2 substrate binding. The invention also features ACE-2 bindingpolypeptide specific antibodies that inhibit binding of the ACE-2binding polypeptide to ACE-2 or ACE-2 binding to an ACE-2 substrate. Inspecific embodiments, antibodies are provided that inhibit ACE-2activity or ACE-2 substrate activity by at least 95%, at least 90%, atleast 85%, at least 80%, at least 75%, at least 70%, at least 60%, or atleast 50% of the activity in absence of the antibody. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra).

[0621] The antibodies of the present invention may be used, for purposesincluding, but not limited to, purify, detect, and target the ACE-2binding polypeptides of the present invention, including both in vitroand in vivo diagnostic and therapeutic methods. For example, theantibodies have use in immunoassays for qualitatively and quantitativelymeasuring levels of ACE-2 in biological samples. See, e.g., Harlow etal., Antibodies: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor 1988).

[0622] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugated) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396 387.

[0623] The antibodies of the invention include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to, specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0624] The antibodies of the present invention may be generated by anysuitable method known in the art. Polyclonal antibodies to anantigen-of-interest can be produced by various procedures well known inthe art. For example, a polypeptide of the invention can be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc. to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

[0625] According to certain embodiments of the invention, multivalentACE-2 binding polypeptides are administered to the host animal.Multivalent ACE-2 binding polypeptide complexes may be prepared usingtechniques and materials known in the art such as, for example, bycross-linking the polypeptide to a carrier protein (e.g., bovine serumalbumin (BSA), human albumin, keyhole limpet hemocyanin (KLH), orsuccinylated KLH) by use of conventional cross-linking reagents.

[0626] In specific embodiments multivalent ACE-2 binding polypeptides ofthe invention are administered in the form of multiple antigen peptides(MAP) (Tam, J. Imm. Meth., 124:53-61 (1989); Tam, Proc. Natl. Acad. Sci.USA, 85:5409-5413 (1988)). In this form, the multivalent ACE-2 bindingpolypeptide is synthesized on a branching lysyl matrix using solid-phasepeptide synthesis methods. Recognition units in the form of MAP may beprepared by methods known in the art (Tam, 1989, supra; Tam, 1988,supra), or, for example, by a stepwise solid-phase procedure on MAPresins (Applied Biosystems), utilizing methodology established by themanufacturer. MAP peptides may be synthesized comprising (ACE-2 bindingpolypeptide)₂ Lys₁, (ACE-2 binding polypeptide)₄ Lys₃, (ACE-2 bindingpolypeptide)₈ Lys₇ or more levels of branching.

[0627] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor 1988); Hammerling et al., in MonoclonalAntibodies and T-Cell Hybridomas (Elsevier, NY 1981), pp. 563-681 (saidreferences incorporated by reference in their entireties). The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced.

[0628] A “monoclonal antibody” may comprise, or alternatively consistof, two proteins, i.e., a heavy and a light chain.

[0629] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Degradation). In anon-limiting example, mice can be immunized with a polypeptide of theinvention or a cell expressing such peptide. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theAmerican Type Culture Collection (ATCC), to form hybridoma cells.Hybridomas are selected and cloned by limited dilution. The hybridomaclones are then assayed by methods known in the art for cells thatsecrete antibodies capable of binding a polypeptide of the invention.Ascites fluid, which generally contains high levels of antibodies, canbe generated by immunizing mice with positive hybridoma clones.

[0630] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with mycloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0631] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)₂ fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0632] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles that carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen-binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods, 182:41-50(1995); Ames et al., J. Immunol. Methods, 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol., 24:952-958 (1994); Persic etal., Gene, 187 9-18 (1997); Burton et al., Advances in Immunology,57:191-280 (1994); PCT international application No. PCT/GB91/01134; PCTpublications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

[0633] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869(1992); and Sawai et al., AJRI, 34:26-34 (1995); and Better et al.,Science, 240:1041-1043 (1988) (said references incorporated herein byreference in their entireties).

[0634] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991);Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerraet al., Science, 240:1038-1040 (1988). For some uses, including in vivouse of antibodies in humans and in vitro detection assays, it may bepreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science, 229:1202(1985); Oi et al., BioTechniques, 4:214 (1986); Gillies et al., J.Immunol. Methods, 125:191-202 (1989); U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816397, which are incorporated herein by reference intheir entirety. A humanized antibody is an antibody molecule made usingone or more complementarity determining regions (CDRs) from a non-humanspecies antibody that binds the desired antigen and framework regionsfrom a human immunoglobulin molecule. Often, framework residues in thehuman framework regions will be substituted with the correspondingresidue from the CDR donor antibody to alter, preferably improve,antigen binding. These framework substitutions are identified by methodswell known in the art, e.g., by modeling of the interactions of the CDRand framework residues to identify framework residues important forantigen binding and sequence comparison to identify unusual frameworkresidues at particular positions. (See, e.g., Queen et al., U.S. Pat.No. 5,585,089; Riechmann et al., Nature, 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239 400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592 106; EP 519 596; Padlan, Molecular Immunology, 28(4/5):489-498(1991); Studnicka et al., Protein Engineering, 7(6):805-814 (1994);Roguska. et al., Proc. Natl. Acad. Sci. USA, 91:969-973 (1994)), andchain shuffling (U.S. Pat. No. 5,565,332).

[0635] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

[0636] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from theimmunized,-transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg and Huszar, Int. Rev. Immunol., 13:65-93 (1995).For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047;WO 96/34096; WO 96/33735; European Patent 598 877; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; 5,885,793; 5,916,771; and 5,939,598, each of which isincorporated by reference herein in its entirety. In addition, companiessuch as Abgenix, Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.)can be engaged to provide human antibodies directed against a selectedantigen using technology similar to that described above.

[0637] Completely human antibodies that recognize a selected epitope canbe generated using a technique referred to as “guided selection.” Inthis approach, a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (See, Jespers et al., Bio/technology,12:899-903 (1988).)

[0638] Further, antibodies to the ACE-2 binding polypeptides of theinvention can, in turn, be utilized to generate anti-idiotype antibodiesthat “mimic” polypeptides of the invention using techniques well knownto those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J.,7(5):437-444 (1989) and Nissinoff, J. Immunol., 147(8):2429-2438(1991).) For example, antibodies which bind to and competitively inhibitthe binding of ACE-2 binding polypeptide to ACE-2 can be used togenerate anti-idiotypes that “mimic” the ACE-2/ACE-2 binding polypeptidebinding domain and, as a consequence, bind to and neutralize or enhanceACE-2 binding to an ACE-2 substrate (e.g., angiotensin, bradykinin,tachykinin, endothelin, neurotensin, or Substance P). Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to bind ACE-2 and/or neutralize or enhance ACE-2mediated acitivity. In a specific embodiment, anti-idiotypic antibodiescan be used to -bind ACE-2, and thereby block its biological activity.In another specific embodiment, anti-idiotypic antibodies can be used tobind ACE-2, and thereby enhance its biological activity (e.g., viamultimerization of ACE-2).

[0639] Polynucleotides Encoding Antibodies

[0640] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent hybridization conditions, e.g., as defined supra, topolynucleotides that encode an antibody, preferably, that specificallybinds to ACE-2 or an ACE-2 binding polypeptide of the invention.

[0641] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques, 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0642] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method known in the art.

[0643] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2dEd. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1990) andCurrent Protocols in Molecular Biology, Ausubel et al., eds. (John Wiley& Sons, NY 1993), which are both incorporated by reference herein intheir entireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

[0644] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well known in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol., 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds ACE-2 or an ACE-2binding polypeptide of the invention. Preferably, as discussed supra,one or more amino acid substitutions may be made within the frameworkregions, and, preferably, the amino acid substitutions improve bindingof the antibody to its antigen. Additionally, such methods may be usedto make amino acid substitutions or deletions of one or more variableregion cysteine residues participating in an intrachain disulfide bondto generate antibody molecules lacking one or more intrachain disulfidebonds. Other alterations to the polynucleotide are encompassed by thepresent invention and within the skill of the art.

[0645] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:851-855(1984); Neuberger et al., Nature, 312:604-608 (1984); Takeda et al.,Nature, 314:452-454 (1985)) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.As described supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine antibody and a humanimmunoglobulin constant region, e.g., humanized antibodies.

[0646] Alternatively, techniques described for the production of singlechain antibodies.(U.S. Pat. No. 4,946,778; Bird, Science, 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988);and Ward et al., Nature, 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science, 242:1038-1041 (1988)).

[0647] Methods of Producing Antibodies

[0648] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0649] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody or portionthereof (preferably containing the heavy or light chain variable domain)of the invention has been obtained, the vector for the production of theantibody molecule may be produced by recombinant DNA technology usingtechniques well known in the art. Thus, methods for preparing a proteinby expressing a polynucleotide containing an antibody-encodingnucleotide sequence are described herein. Methods which are well knownto those skilled in the art can be used to construct expression vectorscontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors may includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT publication WO 86/05807; PCT publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

[0650] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0651] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene, 45:101 (1986); Cockett et al., Bio/Technology,8:2 (1990)).

[0652] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J., 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res., 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.,24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0653] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0654] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. See, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359(1984). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see, Bittner et al.,Methods in Enzymol., 153:51-544 (1987)).

[0655] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,NSO, 293, 3T3, W138, and in particular, breast cancer cell lines suchas, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammarygland cell line such as, for example, CRL7030 and Hs578Bst.

[0656] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0657] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell, 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell, 22:817 (1980))genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc.Natl. Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418; Wuand Wu, Biotherapy, 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.Toxicol., 32:573-596 (1993); Mulligan, Science, 260:926-932 (1993); andMorgan and Anderson, Ann. Rev. Biochem., 62:191-217 (1993); May, 1993,TIB TECH 11(5):155-215); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology may be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Current Protocols in Molecular Biology, Ausubel et al.,eds. (John Wiley & Sons, NY 1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual (Stockton Press, NY 1990); and CurrentProtocols in Human Genetics, Dracopoli et al., eds. (John Wiley & Sons,NY 1994), Chapters 12 and 13; Colberre-Garapin et al., J. Mol. Biol.,150:1 (1981), which are incorporated by reference herein in theirentireties.

[0658] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol., 3:257(1983)).

[0659] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature, 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA, 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0660] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0661] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalent and non-covalentconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanACE-2 binding polypeptides of the present invention. For example,antibodies may be used to target the polypeptides of the presentinvention to particular cell types, either in vitro or in vivo, byfusing or conjugating the polypeptides of the present invention toantibodies specific for particular cell surface receptors. Antibodiesfused or conjugated to the polypeptides of the present invention mayalso be used in in vitro immunoassays and purification methods usingmethods known in the art. See e.g., Harbor et al., supra, and PCTpublication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett.,39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., Proc. Natl.Acad. Sci. USA, 89:1428-1432 (1992); Fell et al., J. Immunol.,146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0662] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307 434; EP 367 166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA, 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0663] As discussed, supra, the polypeptides corresponding to an ACE-2binding polypeptide of the invention may be fused or conjugated to theabove antibody portions to increase the in vivo half life of thepolypeptides or for use in immunoassays using methods known in the art.Further, the ACE-2 binding polypeptides of the invention may be fused orconjugated to the above antibody portions to facilitate purification.One reported example describes chimeric proteins consisting of the firsttwo domains of the human CD4-polypeptide and various domains of theconstant regions of the heavy or light chains of mammalianimmunoglobulins. (EP 394 827; Traunecker et al., Nature, 331:84-86(1988). The polypeptides of the present invention fused or conjugated toan antibody having disulfide-linked dimeric structures (due to the IgG)may also be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.(Fountoulakis et al., J. Biochem., 270:3958-3964 (1995)). In many cases,the Fc part in a fusion protein is beneficial in therapy and diagnosis,and thus can result in, for example, improved pharmacokinetic properties(see, EP-A-232 262). Alternatively, deleting the Fc part after thefusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition, 8:52-58 (1995); Johanson et al., J. Biol. Chem.,270:9459-9471 (1995).

[0664] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA, 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell,37:767 (1984)) and the “flag” tag.

[0665] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic-, group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

[0666] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0667] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha,TNF-beta, AIM I (See, PCT publication WO 97/33899), AIM II (See, PCTpublication WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol.,6:1567-1574 (1994)), VEGI (See, PCT publication WO 99/23105), CD40Ligand, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0668] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al., eds. (Alan R. Liss, Inc. 1985), pp.243-56; Hellstrom et al., “Antibodies For Drug Delivery”, in ControlledDrug Delivery (2nd Ed.), Robinson et al., eds. (Marcel Dekker, Inc.1987), pp. 623-53; Thorpe, “Antibody Carriers Of Cytotoxic Agents InCancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al., eds., pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody in Cancer Therapy”, in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al., eds. (Academic Press1985), pp. 303-16; and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982).

[0669] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the ACE-2binding polypeptide. Such solid supports include, but are not limitedto, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride or polypropylene.

[0670] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0671] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

[0672] Assays for Antibody Binding

[0673] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Current Protocols inMolecular Biology, Ausubel et al., eds. (John Wiley & Sons, NY 1993),which is incorporated by reference herein in its entirety). Exemplaryimmunoassays are described briefly below (but are not intended by way oflimitation).

[0674] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. (JohnWiley & Sons, NY 1993) at 10.16.1.

[0675] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Current Protocols in Molecular Biology,Ausubel et al., eds. (John Wiley & Sons, NY 1993) at 10.8.1.

[0676] ELISAs comprise preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds. (John Wiley& Sons, NY 1993) at 11.2.1.

[0677] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., ³H or ¹²⁵I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., ³H or ¹²⁵I) in the presence of increasingamounts of an unlabeled second antibody.

[0678] Therapeutic Uses of Antibodies

[0679] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the diseases, disorders, or conditions disclosedherein. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant ACE-2expression and/or activity, including, but not limited to, any one ormore of the diseases, disorders, or conditions described herein.

[0680] The treatment and/or prevention of diseases, disorders, orconditions associated with aberrant expression and/or activity of ACE-2or an ACE-2 substrate includes, but is not limited to, alleviatingsymptoms associated with those diseases, disorders or conditions. Theantibodies of the invention may also be used to target and kill cellsexpressing ACE-2 on their surface and/or cells having ACE-2 bound totheir surface. This targeting may be the result of binding of theantibody to ACE-2 binding polypeptides of the invention that have beencoadministered, or alternatively, the result of direct binding of theantibody to ACE-2. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0681] Non-limiting examples of the ways in which the antibodies of thepresent invention may be used therapeutically includes binding ACE-2binding polypeptides of the present invention that have beencoadministered in order to bind or neutralize ACE-2, or by directcytotoxicity of the antibody, e.g., as mediated by complement (CDC) orby effector cells (ADCC). ACE-2 binding polypeptides and anti-ACE-2binding polypeptide antibodies may be administered either locally orsystemically. Some of these approaches are described in more detailbelow. Armed with the teachings provided herein, one of ordinary skillin the art will know how to use the antibodies of the present inventionfor diagnostic, monitoring or therapeutic purposes without undueexperimentation.

[0682] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0683] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents,antibiotics, and immunoglobulin). Generally, administration of productsof a species origin or species reactivity (in the case of antibodies)that is the same species as that of the patient is preferred. Thus, in apreferred embodiment, human antibodies, fragments derivatives, analogs,or nucleic acids, are administered to a human patient for therapy orprophylaxis.

[0684] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides of thepresent invention, fragments or regions thereof, for both immunoassaysdirected to and therapy of disorders related to polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or K_(D) less than 5×10⁻⁵ M,10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

[0685] Demonstration of Therapeutic or Prophylactic Activity ofAntibodies

[0686] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0687] Therapeutic and/or Prophylactic Administration and Composition

[0688] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred embodiment, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0689] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0690] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem., 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0691] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0692] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science,249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler, eds. (Liss,New York 1989), pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0693] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:201(1987); Buchwald et al., Surgery, 88:507 (1980); Saudek et al., N. Engl.J. Med., 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer andWise, eds. (CRC Press, Boca Raton, Fla. 1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball,eds. (Wiley, New York 1984); Ranger and Peppas, J. Macromol. Sci. Rev.Macromol. Chem., 23:61 (1983); see also Levy et al., Science, 228:190(1985); During et al., Ann. Neurol., 25:351 (1989); Howard et al., J.Neurosurg., 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,in Medical Applications of Controlled Release, Langer and Wise, eds.(CRC Press, Boca Raton, Fla. 1974), vol. 2, pp. 115-138 (1984)).

[0694] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0695] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA, 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0696] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules; powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co.,1990). Such compositions will contain a therapeutically effective amountof the compound, preferably in purified form, together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. The formulation should suit the mode of administration.

[0697] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0698] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0699] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0700] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0701] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0702] Diagnosis and Imaging

[0703] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to an ACE-2 binding polypeptide of interest can beused for diagnostic purposes to detect, diagnose, or monitor diseasesand/or disorders associated with the aberrant expression and/or activityof ACE-2. The invention provides for the detection of aberrantexpression of ACE-2, comprising (a) contacting cells or body fluid withan ACE-2 binding polypeptide; (b) assaying the expression of ACE-2 incells or body fluid of an individual using one or more antibodiesspecific to the ACE-2 binding polypeptide and (c) comparing the level ofACE-2 expression with a standard ACE-2 expression level, whereby anincrease or decrease in the assayed ACE-2 expression level compared tothe standard expression level is indicative of aberrant expression.

[0704] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) contacting cells or body fluid with an ACE-2binding polypeptide; (b) assaying the expression of ACE-2 in cells orbody fluid of an individual using one or more antibodies specific to theACE-2 binding polypeptide of interest and (c) comparing the level ofACE-2 expression with a standard ACE-2 expression level, whereby anincrease or decrease in the assayed ACE-2 expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount ofACE-2 in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0705] Antibodies of the invention can be used to assay ACE-2 proteinlevels in a biological sample using or routinely modifying classicalimmunohistological methods known to those of skill in the art (e.g., seeJalkanen et al., J. Cell. Biol., 101:976-985 (1985); Jalkanen et al., J.Cell. Biol., 105:3087-3096 (1987)). Other antibody-based methods usefulfor detecting protein gene expression include immunoassays, such as theenzyme linked immunosorbent assay (ELISA) and the radioimmunoassay(RIA). Suitable antibody assay labels are known in the art and includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc,^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0706] Techniques known in the art may be applied to label antibodies ofthe invention. Such techniques include, but are not limited to, the useof bifunctional conjugating agents (see, e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).

[0707] One embodiment of the invention is the detection and diagnosis ofa disease or disorder associated with aberrant expression of ACE-2 in ananimal, preferably a mammal and most preferably a human. In oneembodiment, diagnosis comprises: (a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to ACE-2(e.g., an ACE-2 binding polyptide of the invention) or whichspecifically binds to a molecule that specifically binds to ACE-2 (e.g.,an anti-ACE-2 binding polypeptide antibody of the invention); (b)waiting for a time interval following the administering for permittingthe labeled molecule to preferentially concentrate at sites in thesubject where the polypeptide is expressed (and for unbound labeledmolecule to be cleared to background level); (c) determining backgroundlevel; and (d) detecting the labeled molecule in the subject, such thatdetection of labeled molecule above the background level indicates thatthe subject has a particular disease or disorder associated withaberrant expression of the polypeptide of interest. Background level canbe determined by various methods including, comparing the amount oflabeled molecule detected to a standard value previously determined fora particular system.

[0708] It will be understood by those skilled in the art that the sizeof the subject and the imaging system used will determine the quantityof imaging moiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ⁹⁹ mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specificpolypeptide. In vivo tumor imaging is described in S. W. Burchiel etal., “Immunopharmacokinetics of Radiolabeled Antibodies and TheirFragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection ofCancer, S. W. Burchiel and B. A. Rhodes, eds. (Masson Publishing Inc.1982).

[0709] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0710] In a further embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisorder, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc. andcomparing the results.

[0711] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include but are not limited to computed tomography (CT), wholebody scan such as position emission tomography (PET), magnetic resonanceimaging (MRI), and sonography.

[0712] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0713] Antibody Kits

[0714] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention comprisetwo or more antibodies (monoclonal and/or polyclonal) that recognize thesame and/or different sequences or regions of a polypeptide according tothe invention. In another specific embodiment, the kits of the presentinvention contain a means for detecting the binding of an antibody to apolypeptide of interest (e.g., the antibody may be conjugated to adetectable substrate such as a fluorescent compound, an enzymaticsubstrate, a radioactive compound or a luminescent compound, or a secondantibody which recognizes the first antibody may be conjugated to adetectable substrate).

[0715] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0716] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0717] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0718] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

[0719] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated protein(s).

[0720] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0721] In another specific embodiment, any of the antibodies listedabove are conjugated to a toxin or a label (as described supra). Suchconjugated antibodies are used to kill a particular population of cellsor to quantitate a particular population of cells. In a preferredembodiment, such conjugated antibodies are used to kill smooth musclecells expressing ACE-2 on their surface. In another preferredembodiment, such conjugated antibodies are used to quantitate smoothmuscle cells expressing ACE-2 on their surface. In a further preferredembodiment, such conjugated antibodies are used to kill endothelialcells expressing ACE-2 on their surface. In a further preferredembodiment, such conjugated antibodies are used to quantitateendothelial cells expressing ACE-2 on their surface.

[0722] In another specific embodiment, any of the antibodies listedabove are conjugated to a toxin or a label (as described supra). Suchconjugated antibodies are used to kill a particular population of cellsor to quantitate a particular population of cells. In a preferredembodiment, such conjugated antibodies are used to kill smooth musclecells expressing the membrane-bound form of ACE-2. In another preferredembodiment, such conjugated antibodies are used to quantitate smoothmuscle cells expressing the membrane-bound form of ACE-2. In a furtherpreferred embodiment, such conjugated antibodies are used to killendothelial cells expressing the membrane-bound form of ACE-2. In afurther preferred embodiment, such conjugated antibodies are used toquantitate endothelial cells expressing the membrane-bound form ofACE-2.

[0723] The antibodies of the invention also have uses as therapeuticsand/or prophylactics which include, but are not limited to, regulationof vasoconstriction, as an analgesic agent, regulation of smooth musclecell proliferation, and in activating cells or blocking cell activationand/or killing cell lineages that express the membrane bound form ofACE-2 on their cell surfaces (e.g., to treat, prevent, and/or diagnoseatherosclerosis, restenosis, and other diseases or conditions). In aspecific embodiment, the antibodies of the invention fix complement. Inother specific embodiments, as further described herein, the antibodiesof the invention (or fragments thereof) are associated with heterologouspolypeptides or nucleic acids (e.g. toxins, such as, compounds that bindand activate endogenous cytotoxic effecter systems, and radioisotopes;and cytotoxic prodrugs).

[0724] As discussed above, antibodies to the ACE-2 binding polypeptidesof the invention can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” the ACE-2 binding polypeptide, using techniqueswell known to those skilled in the art. (See, e.g., Greenspan & Bona,FASEB J., 7(5):437-444 (1989), and Nissinoff, J. Immunol.,147(8):2429-2438 (1991)). For example, antibodies which bind to ACE-2binding polypeptides and competitively inhibit ACE-2,/ACE-2 bindingpolypeptide binding can be used to generate anti-idiotypes that “mimic”the ACE-2 binding polypeptide/ACE-2 binding domain and, as aconsequence, bind to and, for example, neutralize ACE-2. Suchneutralizing anti-idiotypes or Fab fragments of such anti-idiotypes canbe used in therapeutic regimens to neutralize ACE-2. For example, suchanti-idiotypic antibodies can be used to bind ACE-2 and thereby blockACE-2 mediated vasoconstriction.

EXAMPLES

[0725] Isolation of ACE-2 binding polypeptides and their use inaccordance with this invention will be further illustrated below. Thespecific parameters included in the following examples are intended toillustrate the practice of the invention, and they are not presented toin any way limit the scope of the invention.

Example 1 Screening of Phage Display Libraries

[0726] The specific polypeptides according to this invention wereselected from screening eight phage display libraries. Six of thelibraries each displayed a short, variegated exogenous peptide loop of6, 7, 8, 9, 10, or 12 amino acids on the surface of M13 phage, at theamino terminus of protein III. The libraries are designated TN6/6(having a potential 3.3×10¹² amino acid sequence diversity); TN7/4(having a potential 1.2×10¹⁴ amino acid sequence diversity), TN8/9(having a potential 2.2×10¹⁵ amino acid sequence diversity), TN9/4(having a potential 4.2×10¹⁶ amino acid sequence diversity, TN10/9(having a potential 3.0×10¹⁶ amino acid sequence diversity), and TN12/1(having a sequence diversity of 4.6×10¹⁹).

[0727] The TN6/6 library was constructed to display a singlemicroprotein binding loop contained in a 12-amino acid template. TheTN6/6 library utilized a template sequence ofXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO:153). Theamino acids at positions 2, 3, 5, 6, 7, 8, 10, and 11 of the templatewere varied to permit any amino acid except cysteine (C). The aminoacids at positions 1 and 12 of the template were varied to permit anyamino acid except cysteine (C), glutamic acid (E), isoleucine (I),Lysine (K), methionine (M), and threonine (T).

[0728] The TN7/4 library was constructed to display a singlemicroprotein binding loop contained in a 13-amino acid template. TheTN7/4 library utilized a template sequence ofXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO:154). Theamino acids at amino acid positions 1, 2, 3, 5, 6, 7, 8, 9, 11, 12, and13 of the template were varied to permit any amino acid except cysteine(C).

[0729] The TN8/9 library was constructed to display a singlemicroprotein binding loop contained in a 14-amino acid template. TheTN8/9 library utilized a template sequence ofXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO:155).The amino acids at positions 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 13, and 14in the template were varied to permit any amino acid except cysteine(C).

[0730] The TN9/4 library was constructed to display a singlemicroprotein binding loop contained in an 15-amino acid template. TheTN9/1 library utilized a template sequenceXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ ID NO:156). The amino acids at positions 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 13,14 and 15 in the template were varied to permit any amino acid exceptcysteine (C).

[0731] The TN10/9 library was constructed to display a singlemicroprotein binding loop contained in a 16-amino acid template. TheTN10/9 library utilized a template sequenceXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa (SEQ IDNO:157). The amino acids at positions 1, 2, 15, and 16 in the templatewere varied to permit any amino acid selected from a group of 10 aminoacids: D, F, H, L, N, P, R, S, W, or Y). The amino acids at positions 3and 14 in the template were varied to permit any amino acid selectedfrom a group of 14 amino acids: A, D, F, G, H, L, N, P, Q, R, S, V, W,or Y). The amino acids at positions 5, 6, 7, 8, 9, 10, 11, and 12 in thetemplate were varied to permit any amino acid except cysteine (C).

[0732] The TN12/1 library was constructed to display a singlemicroprotein binding loop contained in an 18-amino acid template. TheTN12/1 library utilized a template sequenceXaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Xaa(SEQ ID NO:158). The amino acids at position 1, 2, 17, and 18 in thetemplate were varied to permit any amino acid selected from a group of12 amino acids: A, D, F, G, H, L, N, P, R, S, W, or Y). The amino acidsat positions 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 16 were varied topermit any amino acid except cysteine (C).

[0733] We also endeavored to select ACE-2 binding polypeptides from twocommercially available linear phage display libraries, designated PhD 7and PhD 12, respectively (New England Biolabs). The PhD 7 librarydisplays a linear random-sequence 7-mer; the PhD 12 libary displays arandom-sequence 12-mer. No ACE-2 binding phage were isolated from thesetwo linear libraries.

[0734] The libraries were selected against a FLAG-tagged ACE-2 target,which was immobilized to streptoavidin-coated paramagnetic beads(Seradyn, Ramsey, MN) via biotinylated anti-FLAG antibody (M2 antibody,Sigma, St. Louis, Mo.). Before selection against the target, thelibraries were depleted 5 times with FLAG peptide/anti-FLAGantibody-immobilized beads, to remove binders to the streptoavidinbeads, the anti-FLAG antibody and the FLAG peptide. The depletedlibraries were incubated with 6 μg FLAG-ACE-2 (obtained from HumanGenome Sciences, Inc.) in solution for 1 hour at room temperature (RT),and then incubated with anti-FLAG antibody-immobilized beads for 1 hour.The beads were washed 7 times with PBS, 0.2% Tween 20 (PBST) to removeunbound phage. After washing, the bound phage were eluted by introducingfree FLAG peptide (100 μg/ml). Eluted phage were amplified, andunderwent two more similar rounds of selection. In round 1, the sixconstrained peptide libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, andTN12/1, were selected separately. To accelerate the selectionprocedures, in the subsequent rounds of selection, these six librarieswere combined into two pools: TN6/6, TN7/4, and TN8/9 were combined toform pool A, whereas TN9/4, TN10/9, and TN12/1 were combined to formpool B. The two linear peptide libraries, PhD7 and PhD 12 (New EnglandBioLabs) were combined together at round 1.

[0735] Phage enriched from the third round of selection were screened byELISA for strong ACE-2 binders. Immulon 2 96-well plates (Dynatech) werecoated with streptavidin for 1 hour at 37° C. and subsequently coatedwith anti-FLAG antibody for 1 hour at room temperature. Half of theplates were further coated with FLAG-ACE-2 as the target plates, and theother half of the plates were coated with FLAG peptide as backgroundplates. The amount of each protein or peptide coated was 100 ng perwell. The coated plates were then incubated for 1 hour at roomtemperature with 1:2 diluted overnight phage cultures that were made byinoculating phage from individual plaques into bacterial host cells.After being washed 7 times with PBST, the plates were incubated withHRP-conjugated anti-M13 antibody (Pharmacia) for 1 hour at roomtemperature, washed 5 times, developed with TMB peroxidase substratesolution (Kirkegaad & Perry Laboratories, Gaithersburg, M), and read at630 nm on an ELISA plate reader.

[0736] DNA sequences encoding peptides from positive phage binders wereamplified by PCR and sequenced by automatic sequencing. A number ofACE-2-binding polypeptides were identified, with ACE-2 binders beingisolated from five of the cyclic peptide libraries screened (i.e., allexcept TN9/4). Analysis of recurring amino acid sequences among theACE-2 binding polypeptides revealed a series of polypeptide “families”exhibiting common core structures: Sequence Family I SEQ ID NO:        N R E C H A L F C M D F 20         S P T C R A L F C V D F 21        S E N C Q A L F C V D F 22         S P T C R A L F C V D F 23        L E M C E A L F C V E F 24         N P E C G A L F C M E F 25        D F G C N A M F C V E F 26         D Q N C F A M Y C F E F 27 ND Y C T V F T G A L F C L D F 28 P N Q C G V D I W A L F C V D F 29Sequence Family II SEQ ID NO:         E G N C F L I G P W C F E F 30        E G N C F L I G P W C F E F 31         H I E C E E W G Y W C I EM 32         W E D C L W I G M M C V E F 33         Y E D C I G H A L FC M T F 34         D D K C F G W A H F C F D F 35         G G Q C G T SY L F C I D F 36         Y S G C A D M Y M F C I D F 37         G G Q CG T S Y L F C I D F 38         K F E C M P S S L F C V D F 39 D D Y C FN I S S Y S Y C F D F 40 L H D C F I Y A D Y E Y C F D F 41 N H H C L EF S S F E Y C F D F 42 D N L C M S G G S F D Y C F D F 43 S D Y C V G NN A V T Y C F D F 44 N L D C I Y L Q N H S Y C F D F 45 D D D C M M L PL T M F C F D F 46 Y D N C L G L A N L N F C F D F 47 H L D C Y N L V DN M F C F D F 48 N W N C L G T N E L Q F C L D F 49 Y F A C T N N D S YL F C L D F 50 Y N F C M L I G E R D Y C L D F 51 D D V C Y S L I M A DY C L D F 52 Y F A C T N N D S Y L F C L D F 53 Sequence Family III SEQID NO: D D M C R W Y P F A S F Y M C L F - 54 D D H C E W A S Y W K W DL C L H D 55 D D V C E N A D F A W L G W C M H F 56 D D D C G W I G F AN F H L C L H G 57 F D D C Q T S W F Q G F W L C I D D 58 F H D C S W GP W G P W E I C T R L 59 S N D C V W L Q F W G G D M C F L P 60 N A D CE W V N F N H V D L C M W N 61 G S D C E W V N F T M F Q M C I S N 62 AW D C E W N L F D S T F F C P G F 63 L Y E C E W K Q F G P V E M C L N F64 H S E C R W E W F G R T M I C M S F 65 S G E C N W Q Q F S G W E I CL R D 66 A Y L C D W I L F D S F E M C L A P 67 P F E C D W G P W T L EM L C G P P 68 Sequence Family IV SEQ ID NO:         R G H C R D S R C MM N A P G 69         R I G C R D S R C N W W A P G 70         R G F C RD S S C S F P 71         R G W C L D S R C K V F 72   F L F C R L A S RD S R C A S P 73   F N P C R L Q S R D S A C R F R 74   F F P C R A L EK D S R C S F F 75   H F S C R L P S L D S R C Q L W 76 Sequence FamilyV SEQ ID NO: N D V C L N D D C V Y G 77 W P T C L T M D C V Y N 78 H Y NC H T N D C V V L 79 H L R C M T S D C I H F 80 Sequence Family VI SEQID NO: W V L C F E W E D C D E K 81 Y E Y C F E W E Q C W E K 82 G I F CF E W E T C Y Q A 83 P Q F C F E W E P C F - - 84 I G F C F E W E V C YE G 85 S T Y C F D W E D C W D E 86 Y D W C F D W E Q C W D Q 87 V G F CF D W E P C D E L 88 M D F C F D W E E C W T N 89 N I F C F D W E P C HF G 90 F E I C F D W E V C H E Q 91 D Y L C F D W E A C W L S 92 Y A M CF D W D E C F L G 93 W?W C F E W E D W C L V E 94 Y Q F C F D W E T T CW L D 95 V Y F C F D W E Q D C D E M 96 F Q L C F D W E E E C E E S 97 WA V C F D W E N - C G D K 98 W Q F C F D W D L N C D L R 99 Y W F C F DW E E D A N G H C G G N 100 F L L C F D W D I D W E Y G C Q H H 101Sequence Family VII SEQ ID NO: Y E E C H W R P M A C S T H 102 W E V C HW A P M M C K H G 103 Y E F C H Y A P Q E C K H M 104 Sequence FamilyVIII SEQ ID NO: ?K E C K F G Y S ?C L A W 105 Q K E C K F G Y P H C L PW 106 Sequence Family IX SEQ ID NO:    E H N C T W W N P C W T T 107   M D H C T W Y Q P C V L K 103    W D H C N W A H P C S R K 109    S DW CGT W N N P C F H Q 110 Sequence Family X SEQ ID NO: R Y L C L P Q R DK P W K F C N W F 111 R L H C K P Q R Q S P W M K C Q H L 112 Y S H C SP L R Y Y P W W K C T Y P 113 L H A C R P V R G D P W W A C T L G 114 GF T C S P I R M F P W F R C D L G 115 F S P C K A L R H S P W W V C P SG 116

[0737] In the foregoing peptide families, the amino acids in bold typeare either invariant at that position or are preferred (i.e., recurrentin multiple sequences) in a position relative to an invariant residue.Analysis of the structures of the above families of ACE-2 bindingpolypeptides revealed the general formulae I to X for ACE-2 bindersdiscussed above (SEQ ID NOs: 1-10).

[0738] Based on the sequence families identified by sequence clusteranalysis, representative peptides from each motif were synthesized forfurther analysis and testing (see Table 1, below). The crude peptideswere ordered from Sigma (St. Louis, Mo.). The peptides were then cleavedfrom resin, purified, oxidized, and lyophilized. The purity of oxidizedpeptides was great than 90%. TABLE 1 Peptide Sequences Synthesized forFurther Testing Pep- tide SEQ Desig- ID nation Library Sequence NO:DX500 TN6 Ac-GSNRECHALFCMDFAPGEGGG-NH2 11 DX501 TN6Ac-GSSPTCRALFCVDFAPGEGGG-NH2 12 DX502 TN6 Ac-GSLEMCEALFCVEFAPGEGGG-NH213 DX503 TN6 Ac-GSDQNCFAMYCFEFAPGEGGG-NH2 14 DX507 TN10Ac-GSNDYCTVFTGALFCLDFAPEGGG-NH2 18 DX514 TN10Ac-GSPNQCGVDIWALFCVDFAPEGGGK-NH2 25 DX504 TN8Ac-AGEGNCFLIGPWCFEFGTEGGG-NH2 15 DX505 TN8 Ac-AGYEDCIGHALFCMTFGTEGGG-NH216 DX508 TN10 Ac-GSYDNCLGLANLNFCFDFAPEGGG-NH2 19 DX509 TN12Ac-GDDDHCEWASYWKWDLCLHDDPEGGG-NH 20 2 DX510 TN12Ac-GDDDDCGWIGFANFHLCLHGDPEGGG-NH 21 2 DX511 TN12Ac-GDPFECDWGPWTLEMLCGPPDPEGGG-NH 22 2 DX524 TN6Ac-GSRIGCRDSRCNWWAPGEGGG-NH2 27 DX525 TN6 Ac-GSRGFCRDSSCSFPAPGEGGG-NH228 DX526 TN6 Ac-GSWPTCLTMDCVYNAPGEGGG-NH2 29 DX527 TN7Ac-AGWVLCFEWEDCDEKGTEGGG-NH2 30 DX528 TN8 Ac-AGVYFCFDWEQDCDEMGTEGGG-NH231 DX529 TN8 Ac-AGWEVCHWAPMMCKHGGTEGGG-NH2 32 DX530 TN8Ac-AGQKECKFGYPHCLPWGTEGGG-NH2 33 DX531 TN8 Ac-AGSDWCGTWNNPCFHQGTEGGG-NH234 DX512 TN12 Ac-GDRLHCKPQRQSPWMKCQHLDPEGGG-NH 23 2 DX513 TN12Ac-GDLHACRPVRGDPWWACTLGDPEGGG-NH 24 2 DX599 TN12Ac-GDRYLCLPQRDKPWKFCNWFDPEGGG-NH 36 2 DX600 TN12Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-NH 37 2 DX601 TN12Ac-GDGFTCSPIRMFPWFRCDLGDPEGGG-NH 38 2 DX602 TN12Ac-GDFSPCKALRHSPWWVCPSGDPEGGG-NH 39 2

[0739] Lead peptide inhibitor candidates were identified from in vitrostudies utilizing peptide M-2195 as a substrate. Assay mixture wasprepared first by pre-incubating (30 minutes, room temperature) theenzyme with the inhibitor (0.45 μg (50 pmoles) ACE-2,0-0.5 μM inhibitorrage, up to 90 μL volume with 100 mM TRIS pH 7.4, 0.1% Tween-20, 0.4%DMSO) in each well, in triplicate, by dispensing volumes using a repeatpipetter (Rainin) into a 96-well clear-bottom back plate (Costar,07-200-590). The layout of the plate was such that M-2195 substrateconcentration were varied by column and inhibitor concentrations werevaried by row. Substrate stock (10 μL of 10× concentration) was added toeach well to be tested in blocks of three vertically down the plate. Thereaction was monitored on a Spectrafluor Plus (Tecan) at an excitationwavelength of 340 nm and an emission wavelength of 400 nm (+/−35 nm)every 30 seconds for a time period of 10 minutes. All data points weretransferred to and analysed by Prism 2.01 software. The results areshown in table 2. TABLE 2 Clone #of Inhibi IC50 Ki SEQ ID DX-No. NameSequence Res. tion μM nM NO: DX-500 A-B5 Ac-GSNRECHALFCMDFAPGEGGG-NH221 + 11 DX-501 A-H2 Ac-GSSPTCRALFCVDFAPGEGGG-NH2 21 + 12 DX-502 A-B6Ac-GSLEMCEALFCVEFAPGEGGG-NH2 21 − 13 DX-503 A-C11Ac-GSDQNCFAMYCFEFAPGEGGG-NH2 21 − 14 DX-504 A-F12Ac-AGEGNCFLIGPWCFEFGTEGGG-NH2 22 − 15 DX-505 A3-E12Ac-AGYEDCIGHALFCMTFGTEGGG-NH2 22 + 16 *DX-506 ACEH10-A6Ac-AGWELCNGVMALFCVEFGTEGGG-NH2 23 n./d. 17 DX-507 ACEH5-H8Ac-GSNDYCTVFTGALFCLDFAPEGGG-NH2 24 − 18 DX-508 ACEH1-C1Ac-GSYDNCLGLANLNFCFDFAPEGGG-NH2 24 + 19 DX-509 ACEH6-D12Ac-GDDDHCEWASYWKWDLCLHDDPEGGG-NH2 26 + 20 DX-510 ACEH4-H9Ac-GDDDDCGWIGFANFHLCLHGDPEGGG-NH2 26 − 21 DX-511 ACEH1-D3Ac-GDPFECDWGPWTLEMLCGPPDPEGGG-NH2 26 + 22 DX-512 B-H7Ac-GDRLHCKPQRQSPWMKCQHLDPEGGG-NH2 26 ++++ 0.06 150 23 DX-513 ACEH2-D8Ac-GDLHACRPVRGDPWWACTLGDPEGGG-NH2 26 ++++ 0.09 150 24 DX-514 ACEH2-A2Ac-GSPNQCGVDIWALFCVDFAPEGGGK-NH2 25 + 25 DX-515 ACEH2-A2Ac-GSPNQCGVDIWALFCVDFAPEGGGK(fitc)-NH2 25 26 DX-524 360c-7-G10Ac-GSRIGCRDSRCNWWAPGEGGG-NH2 21 +++ 0.6 27 DX-525 360c-8-H9Ac-GSRGFCRDSSCSFPAPGEGGG-NH2 21 +++ 1 28 DX-526 360c-7-C3Ac-GSWPTCLTMDCVYNAPGEGGG-NH2 21 + 29 DX-527 360c-7-D4Ac-AGWVLCFEWEDCDEKGTEGGG-NH2 21 − 30 DX-528 360c-2-A12Ac-AGVYFCFDWEQDCDEMGTEGGG-NH2 22 − 31 DX-529 360c-4-E5Ac-AGWEVCHWAPMMCKHGGTEGGG-NH2 22 +++ 0.4 32 DX-530 360c-7-D8Ac-AGQKECKFGYPHCLPWGTEGGG-NH2 22 ++ 30 33 DX-531 360c-8-G11Ac-AGSDWCGTWNNPCFHQGTEGGG-NH2 22 +++ 0.5 34 DX-537 ACEH2-D8Ac-GDLHACRPVRGDPWWACTLGDPEGGGK(fitc)-NH2 26 35 DX-599 ACEH2-F6Ac-GDRYLCLPQRDKPWKFCNWFDPEGGG-NH2 26 ++++ 0.14 36 DX-600 ACEH1-F11Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-NH2 26 ++++ 0.025 37 DX-601 ACEH1-G10Ac-GDGFTCSPIRMFPWFRCDLGDPEGGG-NH2 26 ++++ 0.068 38 DX-602 B-G09Ac-GDFSPCKALRHSPWWVCPSGDPEGGG-NH2 26 ++++ 0.12 39

Example 2 Synthesis of Further ACE-2 Binding Peptides

[0740] Once a promising ACE-2 binding polypeptide has been isolated,improvements to that polypeptide can be made by changing, adding orremoving individual or multiple amino acid residues from thepolypeptide. Amino acid substitutions can be conservative or nonconservative. Conservative amino acid exchanges include, for example,the exchange of aromatic residues (e.g., phenylalanine, tryptophan, andtyrosine) for one another, the exchange of hydrophobic residues (e.g,leucine, isoleucine, and valine) for one another, the exchange of polarresidues (e.g., glutamine and asparagine) for one another, the exchangeof acidic residues (e.g., arginine, lysine, and histidine) for oneanother, and the exchange of small residues (e.g., alanine, serine,threonine, methionine, and glycine) for one another, the exchange ofaromatic residues for one another. Additionally, nonclassical aminoacids, chemical amino acid analogs, or chemically modified classicalamino acids can be introduced as a substitution or addition to an ACE-2binding polypeptide of the invention. Non-classical amino acids include,but are not limited to, the D-isomers of the common amino acids,2,4-diaminobutyric acid (Dbu), 4-aminobutyric acid (bAbu),2-aminobutyric acid (Abu), 6-amino hexanoic acid (epsilon-Ahx),2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib),3-aminopropanoic acid (bAla), ornithine (Orn), norleucine (Nle),norvaline (Nva), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp),sarcosine (MeGly), citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,fluoro-amino acids, designer amino acids such as β-methyl amino acids,Cα-methyl amino acids, Noc-methyl amino acids, and amino acid analogs ingeneral.

Example 3 Biacore Analysis of the Affinity of ACE-2 Binding Polypeptides

[0741] Binding of ACE-2 binding polypeptides to ACE-2, for example, canbe analyzed by BIAcore analysis. Either ACE-2 (or another antigen forwhich one wants to know the affinity of an ACE-2 binding polypeptide) orACE-2 binding polpeptide can be covalently immobilized to a BIAcoresensor chip (CM5 chip) via amine groups usingN-ethyl-N′-(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimidechemistry. Various dilutions of ACE-2 binding polypeptides or ACE-2 (orother antigen for which one wants to know the affinity of an ACE-2binding polypeptide), respectively are flowed over the derivatized CM5chip in flow cells at 15 microlters/min. for a total volume of 50microliters. The amount of bound protein is determined during washing ofthe flow cell with HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mMEDTA, 0.005% surfactant P20). Binding specificty for the protein ofinerest is determined by competition with soluble competitor in thepresence the protein of ineterest.

[0742] The flow cell surface can be regenerated by displacing boundprotein by washing with 20 microliters of 10 mM glycine-HCl, pH 2.3. Forkinetic analysis, the flow cells are tested at different flow rates anddifferent polypetide densities on the CM5 chip. The on-rates andoff-rates can be determined using the kinetic evaluation program inBIAevaluation 3 software.

Example 4 In vitro Screening of ACE-2 Antagonists

[0743] The bioassay for assessing the effects of putative ACE-2antagonists is performed in triplicate in 96 well format by mixing equalvolumes of ACE-2, responder cells, and putative antagonist each of whichis prepared as a 3× stock reagent.

[0744] Endothelial cells of coronary vessels are washed and resuspendedin complete medium (CM) (RPMI 1640 with 10% FBS containing 100 U/mlpenicillin, 100 μg/ml streptomycin, 4 mM glutamine, 5×10E-5 Mbeta-mercaptoethanol) at a concentration of 3×10 e6 cells/mL.Staphylococcus aureus, Cowan I (SAC, CalBiochem) is added to cells at 3×concentration (3×=1:33,333 dilution of stock).

[0745] Meanwhile, eight serial dilutions (3-fold) of potentialantagonists are prepared in CM such that the diluted antagonists are at3× the final concentrations to be tested in the assay. ACE-2 bindingpolypeptides are routinely tested starting at a final concentration of10 g/mL and going down to about 1.5 ng/mL.

[0746] Human rACE-2 is prepared in CM to 3× concentration (3×=300 ng/mL,30 ng/mL, and 3 ng/mL) in CM. Potential inhibitors are routinely testedat several concentrations of ACE-2 to avoid false negatives due tounexpectedly low affinity or antagonist concentration.

[0747] Fifty microliters of diluted antagonist and 50 μL of dilutedACE-2 are added to the putative antagonist dilution series. Cells arethen incubated for 72 hours (37° C., 5% CO₂) in a fully humidifiedchamber. After 72 hrs., the cells are supplemented with 0.5 μCi/well3H-thymidine (e.g., 6.7 Ci/mmol) and incubated for an additional 24hours. Plates are harvested using a Tomtec Cell Harvester and filterscounted in a TopCount Scintillation counter (Packard).

Example 5 Protein Fusions of ACE-2 Binding Polypeptides

[0748] ACE-2 binding polypeptides of the invention are optionally fusedto other proteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of ACE-2 binding polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See, EP A 394 827; Traunecker et al., Nature,331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and albuminincreases the half-life time in vivo. Nuclear localization signals fusedto ACE-2 binding polypeptides can target the protein to a specificsubcellular localization, while covalent heterodimer or homodimers canincrease or decrease the activity of a fusion protein. Fusion proteinscan also create chimeric molecules having more than one function.Finally, fusion proteins can increase solubility and/or stability of thefused protein compared to the non-fused protein. All of the types offusion proteins described above can be made using techniques known inthe art or by using or routinely modifying the following protocol, whichoutlines the fusion of a polypeptide to an IgG molecule.

[0749] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below (SEQ ID NO: 184). These primers also preferably containconvenient restriction enzyme sites that will facilitate cloning into anexpression vector, preferably a mammalian expression vector.

[0750] For example, if the pC4 (Accession No. 209646) expression vectoris used, the human Fc portion can be ligated into the BamHI cloningsite. Note that the 3′ BamHI site should be destroyed. Next, the vectorcontaining the human Fc portion is re-restricted with BamHI, linearizingthe vector, and ACE-2 binding polynucleotide is ligated into this BamHIsite. Note that the polynucleotide is cloned without a stop codon,otherwise a fusion protein will not be produced.

[0751] If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891)

[0752] Human IgG Fc Region: (SEQ ID NO: 150)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 6 Isolation of scFv Molecules Recognizing ACE-2 BindingPolypeptides

[0753] Naturally occuring V-genes isolated from human PBLs areconstructed into a large library of antibody fragments which containreactivities against polypeptides of the present invention to which thedonor may or may not have been exposed (see, e.g., U.S. Pat. No.5,885,793, incorporated herein by reference in its entirety). Rescue ofthe library

[0754] A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO 92/01047. To rescue phage displaying antibody fragments,approximately 10⁹ E. coli harbouring the phagemid are used to inoculate50 ml of 2× TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of thisculture is used to innoculate 50 ml of 2× TY-AMP-GLU, 2×10⁸ TU of Δ gene3 helper phage (M13 Δ gene III, see WO 92/01047) are added and theculture incubated at 37° C. for 45 minutes without shaking and then at37° C. for 45 minutes with shaking. The culture is centrifuged at 4000r.p.m. for 10 minutes and the pellet resuspended in 2 liters of 2× TYcontaining 100 ug/ml ampicillin and 50 ug/ml kanamycin and grownovernight. Phage are prepared as described in WO92/01047.

[0755] M13 Δ gene III is prepared as follows: M13 Δ gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 Δ gene III particles are made by growing the helper phagein cells harboring a pUC19 derivative supplying the wild type gene IIIprotein during phage morphogenesis. The culture is incubated for 1 hourat 37° C. without shaking and then for a further hour at 37° C. withshaking. Cells are pelleted (IEC-Centra 8, 4000 revs/min. for 10 min.),resuspended in 300 ml 2× TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2× TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones). Panning of the library

[0756] Immunotubes (Nunc) are coated overnight in PBS with 4 ml ofeither 100 mg/ml or 10 mg/ml of a polypeptide of the present invention.Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and thenwashed 3 times in PBS. Approximately 1013 TU of phage are applied to thetube and incubated for 30 minutes at room temperature tumbling on anover and under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under and over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage arethen used to infect 10 ml of mid-log E. coli TG1 by incubating elutedphage with bacteria for 30 minutes at 37° C. The E. coli are then platedon TYE plates containing 1% glucose and 100 μg/ml ampicillin. Theresulting bacterial library is then rescued with Δ gene III helper phageas described above to prepare phage for a subsequent round of selection.This process is then repeated for a total of 4 rounds of affinitypurification with tube-washing increased to 20 times with PBS, 0.1%Tween-20 and 20 times with PBS for rounds 3 and 4.

[0757] Characterization of Binders

[0758] Eluted phage from the 3rd and 4th rounds of selection are used toinfect E. coli HB 2151 and soluble scFv is produced (Marks et al., 1991)from single colonies for assay. ELISAs are performed with microtitreplates coated with either 10 pg/ml of the polypeptide of the presentinvention in 50 mM bicarbonate, pH 9.6. Clones positive in ELISA arefurther characterized by PCR fingerprinting (see, e.g., WO 92/01047) andthen by sequencing.

[0759] Additionaly, scFvs may be converted to complete Ig moleculesusing techniques which are commonly known in the art.

Example 7 Production of an Anti-ACE-2 Binding Polypeptide AntibodyHybridoma Technology

[0760] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing ACE-2 binding polypeptides areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of ACE-2binding polypeptide is prepared and purified to render it substantiallyfree of natural contaminants which is then conjugated to a carriermolecule such as keyhole limpet hemocyanin (KLH), suucinylated KLH, orchicken gamma globulin (CGG). Such a preparation is then introduced intoan animal in order to produce polyclonal antisera of greater specificactivity.

[0761] In the most preferred method, the antibodies of the presentinvention are monoclonal antibodies (or ACE-2 protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology. (Kohler et al., Nature, 256:495 (1975); Kohler et al., Eur.J. Immunol., 6:511 (1976); Kohler et al., Eur. J. Immunol., 6:292(1976); Hammerling et al., in Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y. 1981), pp. 563-681.) In general, suchprocedures involve immunizing an animal (preferably a mouse) with ACE-2binding polypeptide or, more preferably, with a secreted ACE-2 bindingpolypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

[0762] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP2/0), availablefrom the ATCC. After fusion, the resulting hybridoma cells areselectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands et al. (Gastroenterology, 80:225-232(1981).) The hybridoma cells obtained through such a selection are thenassayed to identify clones which secrete antibodies capable of bindingthe ACE-2 binding polypeptide.

[0763] Alternatively, additional antibodies capable of binding to ACE-2binding polypeptide can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theACE-2 binding polypeptide-specific antibody can be blocked by ACE-2binding polypeptide. Such antibodies comprise anti-idiotypic antibodiesto the ACE-2 binding protein-specific antibody and can be used toimmunize an animal to induce formation of further ACE-2 bindingpolypeptide-specific antibodies.

[0764] It will be appreciated that Fab and F(ab′)₂ and other fragmentsof the antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments). Alternatively,secreted ACE-2 binding protein-binding fragments can be produced throughthe application of recombinant DNA technology or through syntheticchemistry.

[0765] For in vivo use of antibodies in humans, it may be preferable touse “humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science, 229:1202 (1985); Oi et al., BioTechniques, 4:214 (1986);Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171 496;Morrison et al., EP 173 494; Neuberger et al., WO 86/01533; Robinson etal., WO 87/02671; Boulianne et al., Nature, 312:643 (1984); Neuberger etal., Nature, 314:268 (1985)).

Example 8 Fluorometric Assays for ACE-2

[0766] The activity of ACE-2 was measured utilizing a fluorometric assayin which peptide M-2195 (BACHEM) is used as a substrate. ACE-2 cleavesoff the C-terminal lysine from the peptide with a concomitant increasein fluorescence signal.

[0767] The assays were performed in 96-well clear-bottom black plates(Costar, 07-200-590). In each well, 0.45 μg (5 pmoles) of ACE-2 is addedto 90 μL of an assay solution containing 100 mM TRIS at pH 7.4, 0.1%Tween, and 0.4% DMSO. Substrate stock at 10 μL of 10× concentration isadded to each well (for example, 10 μL of 1 mM stock for a final 100 μMconcentration). The reaction was monitored on a Spectrafluor Plus(Tecan) at an excitation wavelength of 340 nm and an emission wavelengthof 400 nm (+/−35 nm) every 30 seconds up to 10 minutes. All samples weremeasured in triplicate. The data points were transferred to and analysedon Prism 2.01 software.

[0768] Degradation: Identification and Analysis of the Functional Rolesof ACE-2 Inhibitory Peptides

[0769] The effects of graded doses of A1-9 (Fields et al., PeptideResearch 4: 95-101 (1991)) or A1-10 (substrate control) alone or incombination (Tallarida, Drug Synergism and Dose-Effect Data Analysis,Chapman and Hall/CRC, New York (2000), Tallarida et al., Life Science61: PL417-425 (1997)) with angiotensin II were examined on isolatedaortic vascular rings developed tension in response to vasoactivesubstances. Aortas were removed from mature male Sprague-Dawley rats,cleared of connective tissue, and cut into 1 mm rings. Rings weremounted to an isometric force transducer and placed in Krebs-Henseleitsolution bubbled with 95% O2/5% CO₂ at 37° C. Rings were exposed to a 2g preload, relaxed and then exposed to various concentrations ofangiotensin peptides alone or in combination. Following angiotensinpeptide dosing, rings were fully constricted with KCl to determine themaximum degree of vasoconstriction.

[0770] Table 3 shows the maximum developed isometric force (Emax)following treatment with A1-9, angiotensin II or the combination of thetwo over a dose range from 1-30 nM (alone or in combination) in maturerat aortic rings. The maximum tension developed by A1-9 was minimal andalways less than that of angiotensin II. When A1-9 was combined withangiotensin II, the resulting Emax was greater than the sum of theindividual peptide Emax values, suggesting a superadditive orsynergistic interaction. Because A1-9 is virtually devoid of activity,the quantitation of its constrictor-enhancing action on angiotensin IIwas derived from the shift in the angiotensin II dose-effect curve asseen in FIG. 1. The slopes of the dose-effect curves did not differ;however, the relative potency of angiotensin II was increased by afactor of 11.6 (95% confidence limits of 3.02-44.7) beyond that ofsimple addition potentiation, indicative of synergism for thecombination. In control experiments, angniotensin I failed to potentiateangiotensin II-mediated vasoconstriction. This indicates that thesynergy observed between A1-9 and angiotensin II is specific for A1-9and is not a property of the ACE and ACE-2 substrate, angiotensin I.TABLE 3 Al-9 % Max. AII % Max. A1-9 + AII % Max. Experiment DevelopedForce Developed Force Developed Force 1 3.5 22 82 2 0 8.6 18 3 7.5 17 244 6.4 9.9 34 5 3.2 41 48

[0771] To investigate the effects of A1-9 on arterial pressure, A1-9 wasadministered to awake, free-ranging Sprague-Dawley rats via indwellingintravenous and arterial catheters. Intravenous infusion of A1-9 (1-3μg/kg/min) in the awake rat produced a dose-dependent tonic increase inmean arterial pressure to a maximum of approximately 30 mmHg (FIG. 2).Pulse pressure (systolic—diastolic) was also elevated and heart rate wasdecreased in a dose-dependent manner by A1-9 infusion (data not shown).These responses seen with A1-9 were similar in magnitude to thoseinduced by angiotensin II. The observed bradycardia (presumed to bereflexive) in association with the increase in mean arterial and pulsepressure suggests that A1-9 increases vascular tone in vivo, perhapsthrough potentiating the activity of angiotensin II.

[0772] To examine any interactions induced by coadministration of A1-9and angiotensin II, arterial pressure was monitored in the awake ratafter they received either A1-9 (1 μg/kg/min) or saline coinfused withangiotensin II (1-100 ng/kg/min). The submaximal infusion of A1-9potentiated the pressor response elicted by angiotensin II in anadditive fashion (FIG. 3). Angiotensin II coinfused with a controlnonapeptide with no intrinsic hemodynamic activity in awake ratsproduced a dose-effect curve similar to that of angiotensin II alone(data not shown).

[0773] To test further the hypothesis that downregulating ACE-2 activitywould decrease arterial pressure, tagged human ACE-2 was generated inorder to identify potential peptide ACE-2 inhibitors whose sequenceswere derived from phage display. The human ACE-2 cDNA was used astemplate for amplification by PCR of the nucleotides corresponding tothe extracellular domain (M1-S740). The 5′ primer: 5′ATGGATGATCAGCCATCATGTCAAGCTCTTCCTG 3′ (SEQ ID NO:151) and 3′ primer: 5′GTATGCTCTAGATTAGGAAACAGGGGGCTGGTTAG 3′ (SEQ ID NO:152) generate a 2200bp fragment which was digested with BclI and XbaI and cloned into theBamHI and XbaI sites of a baculovirus transfer vector pA2. Following DNAsequence confirmation, the plasmid (A2:Ace-H) was transfected into Sf9cells to generate a recombinant baculovirus (Coleman et al, Gene190:163-170 (1997)). Metabolic labeling was used to confirm the presenceof a novel band of ˜85 Kd corresponding to the human Ace-H protein inconditioned media from Sf9 cells. For protein production, Sf9 cells wereseeded in serum-free media and infected at a multiplicity of infectionof 1-2 with the recombinant baculovirus. Conditioned media washarvested, clarified by filtration, and used for subsequent enzymepurification. Initially, FLAG peptide was attached to streptavidin beadsusing bead-immobilized biotinylated anti-FLAG antibody. Proprietaryphage display libraries were depleted on these beads 5 times to removephages bound to the FLAG peptide. Depleted libraries were incubated withFLAG-ACE-2 and then immobilized on streptavidin beads. The beads werewashed stringently and the bound phage was eluted with FLAG peptide.Eluted phages were amplified and characterized by ELISA using FLAG-ACE-2coated in microtiter plates. Positive binders were sequenced andcollapsed into several families based on amino acid sequences.

[0774] A phage display technology was used to identify families ofpeptide inhibitors of rhACE-2. FLAG-ACE-2 (above) was used for panningpeptides binding to ACE-2. Peptide binders were sequenced and collapsedinto several families based upon amino acid sequences. Several membersfrom the peptide families were synthesized and their inhibitoryactivities were tested using recombinant human-ACE-2. Using M-2195peptide as substrate, multiple inhibitors were identified and the mostactive ACE-2 inhibitor peptide was chosen for further studies. AnotherACE-2 binding peptide that failed to inhibit rhACE-2 was identified,synthesized and used as a control for in vivo experiments. The Ki forthe ACE-2 inhibitor peptide was 250 nM, acting as a mixed-type inhibitorfor ACE-2 when M-2195 was used as a substrate. This peptide alsoinhibited conversion of angiotensin I to A1-9 by ACE-2. The ACE-2inhibitor peptide was specific for ACE-2 and did not inhibit theactivity of ACE. For example, the inhibitor did not affect thehydrolysis of the substrate, hip-his-leu (250 mM) by ACE (1.5 nM), evenat 25 mM. In comparison, the hydrolytic activity of ACE was completelyinhibited by 25 mM captopril. Thus, the ACE-2 inhibitor peptide wasfound to be specific for ACE-2 and blocked conversion of angiotensin Ito A1-9.

[0775] A lead inhibitor peptide candidate identified from in vitrostudies caused a dose-dependent depressor response upon iv bolusadministration in the awake SHR (FIG. 4). As the peptide inhibitor dosewas increased, the magnitude and duration of the depressor responseincreased. This depressor response was characterized by an initialtransient fall in MAP lasting approximately 1-2 min at the lower dosesand approximately 6 min in duration at the 3 mg/kg dose level. Themaximal average depressor response at a dose of 3 mg/kg was 70.5±4.6mmHg from an average MAP of 155±10 mmHg. Transient tachycardia (averagemaximal change between 50-70 bpm at a dose level of 2-3 mg/kg) wasobserved to coincide with the depressor response (data not shown). At nodose level was the MAP or HR altered by the control peptide.

[0776] In terms of cardiovascular function, the expression pattern ofACE-2 suggests a potential role in the regulation of local circulationin the kidney and heart. However, our results indicate that ACE-2 mayplay a role in regulating systemic circulatory homeostasis. A1-9, knownto be produced from ACE-2 mediated catabolism of angiotensin I,potentiated angiotensin II-mediated vasoconstriction in isolated rataortic rings and pressor responses in the awake rat. Although there hasbeen speculation that A1-9 might serve to inhibit ACE and lead tovasodilation (Donoghue, Circulation Research 87:e1-e9 (2000), Snyder etal., The Journal of Biological Chemistry 260:7857-7860 (1985)), thepresent results support the hypothesis that ACE-2 upregulates systemicarterial pressure under conditions where intact cardiovascular reflexesare present. An unlikely possibility is that the observed effects ofA1-9 were due to its conversion to angiotensin II in vivo sincebiochemical and in vivo evidence supports that this mechanism is not amajor contributor to the normal formation of angiotensin II (Johnson etal., Peptides 10:489-492 (1989); Donoghue et al., Circulation Research87:e1-e9 (2000); Oparil et al., Circulation Research 29:682-690 (1971)).

[0777] The identification of a peptide inhibitor of ACE-2 has enabledthe study of the effects of ACE-2 inhibition in a model of spontaneoushypertension in vivo. The current results indicate that the inhibitionof ACE-2 reduces arterial pressure through decreasing circulating and/orlevels of A1-9, an angiotensin II synergizing peptide. Supporting thisis the demonstration that blockade of ACE-2 causes a depressor responsein awake spontaneously hypertensive rats.

[0778] Following the foregoing description, the characteristicsimportant for affinity binding polypeptides permitting detection orseparation of ACE-2 or ACE-2-like polypeptides (ACE-2 target protein) inor from any solution can be appreciated. Additional binding polypeptideembodiments of the invention and alternative methods adapted to aparticular solution or feed stream will be evident from studying theforegoing description. All such embodiments and obvious alternatives areintended to be within the scope of this invention, as defined by theclaims that follow.

[0779] Publications referred to above are hereby incorporated byreference.

1 158 1 10 PRT homo sapiens MISC_FEATURE (2)..(2) X equals any aminoacid 1 Glx Xaa Ala Xaa Xaa Cys Xaa Xaa Phe Glx 1 5 10 2 7 PRT homosapiens MISC_FEATURE (2)..(2) X equals any amino acid 2 Glx Xaa Cys XaaXaa Xaa Glx 1 5 3 17 PRT homo sapiens MISC_FEATURE (2)..(4) X equals anyamino acid 3 Glx Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys 1 5 10 15 Glx 4 11 PRT homo sapiens MISC_FEATURE (3)..(6) X equalsany amino acid 4 Glx Arg Xaa Xaa Xaa Xaa Asp Ser Xaa Cys Glx 1 5 10 5 9PRT homo sapiens MISC_FEATURE (3)..(5) X equals any amino acid 5 Glx CysXaa Xaa Xaa Asp Cys Xaa Glx 1 5 6 7 PRT homo sapiens MISC_FEATURE(4)..(4) X equals any amino acid 6 Glx Cys Phe Xaa Trp Xaa Glx 1 5 7 13PRT homo sapiens MISC_FEATURE (2)..(2) X equals any amino acid 7 Glx XaaGlu Xaa Cys His Xaa Xaa Pro Xaa Xaa Cys Glx 1 5 10 8 15 PRT homo sapiensMISC_FEATURE (9)..(10) X equals any amino acid 8 Glx Lys Glu Cys Lys PheGly Tyr Xaa Xaa Cys Leu Xaa Trp Glx 1 5 10 15 9 12 PRT homo sapiensMISC_FEATURE (2)..(3) X equals any amino acid 9 Glx Xaa Xaa Cys Xaa XaaTrp Xaa Xaa Pro Cys Glx 1 5 10 10 14 PRT homo sapiens MISC_FEATURE(3)..(5) X equals any amino acid 10 Glx Cys Xaa Xaa Xaa Arg Xaa Xaa ProTrp Xaa Xaa Cys Glx 1 5 10 11 21 PRT homo sapiens 11 Gly Ser Asn Arg GluCys His Ala Leu Phe Cys Met Asp Phe Ala Pro 1 5 10 15 Gly Glu Gly GlyGly 20 12 21 PRT homo sapiens 12 Gly Ser Ser Pro Thr Cys Arg Ala Leu PheCys Val Asp Phe Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 20 13 21 PRT homosapiens 13 Gly Ser Leu Glu Met Cys Glu Ala Leu Phe Cys Val Glu Phe AlaPro 1 5 10 15 Gly Glu Gly Gly Gly 20 14 21 PRT homo sapiens 14 Gly SerAsp Gln Asn Cys Phe Ala Met Tyr Cys Phe Glu Phe Ala Pro 1 5 10 15 GlyGlu Gly Gly Gly 20 15 22 PRT homo sapiens 15 Ala Gly Glu Gly Asn Cys PheLeu Ile Gly Pro Trp Cys Phe Glu Phe 1 5 10 15 Gly Thr Glu Gly Gly Gly 2016 21 PRT homo sapiens 16 Gly Ser Asp Gln Asn Cys Phe Ala Met Tyr CysPhe Glu Phe Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 20 17 22 PRT homosapiens 17 Ala Gly Glu Gly Asn Cys Phe Leu Ile Gly Pro Trp Cys Phe GluPhe 1 5 10 15 Gly Thr Glu Gly Gly Gly 20 18 24 PRT homo sapiens 18 GlySer Asn Asp Tyr Cys Thr Val Phe Thr Gly Ala Leu Phe Cys Leu 1 5 10 15Asp Phe Ala Pro Glu Gly Gly Gly 20 19 24 PRT homo sapiens 19 Gly Ser TyrAsp Asn Cys Leu Gly Leu Ala Asn Leu Asn Phe Cys Phe 1 5 10 15 Asp PheAla Pro Glu Gly Gly Gly 20 20 26 PRT homo sapiens 20 Gly Asp Asp Asp HisCys Glu Trp Ala Ser Tyr Trp Lys Trp Asp Leu 1 5 10 15 Cys Leu His AspAsp Pro Glu Gly Gly Gly 20 25 21 26 PRT homo sapiens 21 Gly Asp Asp AspAsp Cys Gly Trp Ile Gly Phe Ala Asn Phe His Leu 1 5 10 15 Cys Leu HisGly Asp Pro Glu Gly Gly Gly 20 25 22 26 PRT homo sapiens 22 Gly Asp ProPhe Glu Cys Asp Trp Gly Pro Trp Thr Leu Glu Met Leu 1 5 10 15 Cys GlyPro Pro Asp Pro Glu Gly Gly Gly 20 25 23 26 PRT homo sapiens 23 Gly AspArg Leu His Cys Lys Pro Gln Arg Gln Ser Pro Trp Met Lys 1 5 10 15 CysGln His Leu Asp Pro Glu Gly Gly Gly 20 25 24 26 PRT homo sapiens 24 GlyAsp Leu His Ala Cys Arg Pro Val Arg Gly Asp Pro Trp Trp Ala 1 5 10 15Cys Thr Leu Gly Asp Pro Glu Gly Gly Gly 20 25 25 25 PRT homo sapiens 25Gly Ser Pro Asn Gln Cys Gly Val Asp Ile Trp Ala Leu Phe Cys Val 1 5 1015 Asp Phe Ala Pro Glu Gly Gly Gly Lys 20 25 26 25 PRT homo sapiens 26Gly Ser Pro Asn Gln Cys Gly Val Asp Ile Trp Ala Leu Phe Cys Val 1 5 1015 Asp Phe Ala Pro Glu Gly Gly Gly Lys 20 25 27 21 PRT homo sapiens 27Gly Ser Arg Ile Gly Cys Arg Asp Ser Arg Cys Asn Trp Trp Ala Pro 1 5 1015 Gly Glu Gly Gly Gly 20 28 21 PRT homo sapiens 28 Gly Ser Arg Gly PheCys Arg Asp Ser Ser Cys Ser Phe Pro Ala Pro 1 5 10 15 Gly Glu Gly GlyGly 20 29 21 PRT homo sapiens 29 Gly Ser Trp Pro Thr Cys Leu Thr Met AspCys Val Tyr Asn Ala Pro 1 5 10 15 Gly Glu Gly Gly Gly 20 30 21 PRT homosapiens 30 Ala Gly Trp Val Leu Cys Phe Glu Trp Glu Asp Cys Asp Glu LysGly 1 5 10 15 Thr Glu Gly Gly Gly 20 31 22 PRT homo sapiens 31 Ala GlyVal Tyr Phe Cys Phe Asp Trp Glu Gln Asp Cys Asp Glu Met 1 5 10 15 GlyThr Glu Gly Gly Gly 20 32 22 PRT homo sapiens 32 Ala Gly Trp Glu Val CysHis Trp Ala Pro Met Met Cys Lys His Gly 1 5 10 15 Gly Thr Glu Gly GlyGly 20 33 22 PRT homo sapiens 33 Ala Gly Gln Lys Glu Cys Lys Phe Gly TyrPro His Cys Leu Pro Trp 1 5 10 15 Gly Thr Glu Gly Gly Gly 20 34 22 PRThomo sapiens 34 Ala Gly Ser Asp Trp Cys Gly Thr Trp Asn Asn Pro Cys PheHis Gln 1 5 10 15 Gly Thr Glu Gly Gly Gly 20 35 27 PRT homo sapiens 35Gly Asp Leu His Ala Cys Arg Pro Val Arg Gly Asp Pro Trp Trp Ala 1 5 1015 Cys Thr Leu Gly Asp Pro Glu Gly Gly Gly Lys 20 25 36 26 PRT homosapiens 36 Gly Asp Arg Tyr Leu Cys Leu Pro Gln Arg Asp Lys Pro Trp LysPhe 1 5 10 15 Cys Asn Trp Phe Asp Pro Glu Gly Gly Gly 20 25 37 26 PRThomo sapiens 37 Gly Asp Tyr Ser His Cys Ser Pro Leu Arg Tyr Tyr Pro TrpTrp Lys 1 5 10 15 Cys Thr Tyr Pro Asp Pro Glu Gly Gly Gly 20 25 38 26PRT homo sapiens 38 Gly Asp Gly Phe Thr Cys Ser Pro Ile Arg Met Phe ProTrp Phe Arg 1 5 10 15 Cys Asp Leu Gly Asp Pro Glu Gly Gly Gly 20 25 3926 PRT homo sapiens 39 Gly Asp Phe Ser Pro Cys Lys Ala Leu Arg His SerPro Trp Trp Val 1 5 10 15 Cys Pro Ser Gly Asp Pro Glu Gly Gly Gly 20 2540 12 PRT homo sapiens 40 Asn Arg Glu Cys His Ala Leu Phe Cys Met AspPhe 1 5 10 41 12 PRT homo sapiens 41 Ser Pro Thr Cys Arg Ala Leu Phe CysVal Asp Phe 1 5 10 42 12 PRT homo sapiens 42 Ser Glu Asn Cys Gln Ala LeuPhe Cys Val Asp Phe 1 5 10 43 12 PRT homo sapiens 43 Ser Pro Thr Cys ArgAla Leu Phe Cys Val Asp Phe 1 5 10 44 12 PRT homo sapiens 44 Leu Glu MetCys Glu Ala Leu Phe Cys Val Glu Phe 1 5 10 45 12 PRT homo sapiens 45 AsnPro Glu Cys Gly Ala Leu Phe Cys Met Glu Phe 1 5 10 46 12 PRT homosapiens 46 Asp Phe Gly Cys Asn Ala Met Phe Cys Val Glu Phe 1 5 10 47 12PRT homo sapiens 47 Asp Gln Asn Cys Phe Ala Met Tyr Cys Phe Glu Phe 1 510 48 16 PRT homo sapiens 48 Asn Asp Tyr Cys Thr Val Phe Thr Gly Ala LeuPhe Cys Leu Asp Phe 1 5 10 15 49 16 PRT homo sapiens 49 Pro Asn Gln CysGly Val Asp Ile Trp Ala Leu Phe Cys Val Asp Phe 1 5 10 15 50 14 PRT homosapiens 50 Glu Gly Asn Cys Phe Leu Ile Gly Pro Trp Cys Phe Glu Phe 1 510 51 14 PRT homo sapiens 51 Glu Gly Asn Cys Phe Leu Ile Gly Pro Trp CysPhe Glu Phe 1 5 10 52 14 PRT homo sapiens 52 His Ile Glu Cys Glu Glu TrpGly Tyr Trp Cys Ile Glu Met 1 5 10 53 14 PRT homo sapiens 53 Trp Glu AspCys Leu Trp Ile Gly Met Met Cys Val Glu Phe 1 5 10 54 14 PRT homosapiens 54 Tyr Glu Asp Cys Ile Gly His Ala Leu Phe Cys Met Thr Phe 1 510 55 14 PRT homo sapiens 55 Asp Asp Lys Cys Phe Gly Trp Ala His Phe CysPhe Asp Phe 1 5 10 56 14 PRT homo sapiens 56 Gly Gly Gln Cys Gly Thr SerTyr Leu Phe Cys Ile Asp Phe 1 5 10 57 14 PRT homo sapiens 57 Tyr Ser GlyCys Ala Asp Met Tyr Met Phe Cys Ile Asp Phe 1 5 10 58 14 PRT homosapiens 58 Gly Gly Gln Cys Gly Thr Ser Tyr Leu Phe Cys Ile Asp Phe 1 510 59 14 PRT homo sapiens 59 Lys Phe Glu Cys Met Pro Ser Ser Leu Phe CysVal Asp Phe 1 5 10 60 16 PRT homo sapiens 60 Asp Asp Tyr Cys Phe Asn IleSer Ser Tyr Ser Tyr Cys Phe Asp Phe 1 5 10 15 61 16 PRT homo sapiens 61Leu His Asp Cys Phe Ile Tyr Ala Asp Tyr Glu Tyr Cys Phe Asp Phe 1 5 1015 62 16 PRT homo sapiens 62 Asn His His Cys Leu Glu Phe Ser Ser Phe GluTyr Cys Phe Asp Phe 1 5 10 15 63 16 PRT homo sapiens 63 Asp Asn Leu CysMet Ser Gly Gly Ser Phe Asp Tyr Cys Phe Asp Phe 1 5 10 15 64 16 PRT homosapiens 64 Ser Asp Tyr Cys Val Gly Asn Asn Ala Val Thr Tyr Cys Phe AspPhe 1 5 10 15 65 16 PRT homo sapiens 65 Asn Leu Asp Cys Ile Tyr Leu GlnAsn His Ser Tyr Cys Phe Asp Phe 1 5 10 15 66 16 PRT homo sapiens 66 AspAsp Asp Cys Met Met Leu Pro Leu Thr Met Phe Cys Phe Asp Phe 1 5 10 15 6716 PRT homo sapiens 67 Tyr Asp Asn Cys Leu Gly Leu Ala Asn Leu Asn PheCys Phe Asp Phe 1 5 10 15 68 16 PRT homo sapiens 68 His Leu Asp Cys TyrAsn Leu Val Asp Asn Met Phe Cys Phe Asp Phe 1 5 10 15 69 16 PRT homosapiens 69 Asn Trp Asn Cys Leu Gly Thr Asn Glu Leu Gln Phe Cys Leu AspPhe 1 5 10 15 70 16 PRT homo sapiens 70 Tyr Phe Ala Cys Thr Asn Asn AspSer Tyr Leu Phe Cys Leu Asp Phe 1 5 10 15 71 16 PRT homo sapiens 71 TyrAsn Phe Cys Met Leu Ile Gly Glu Arg Asp Tyr Cys Leu Asp Phe 1 5 10 15 7216 PRT homo sapiens 72 Asp Asp Val Cys Tyr Ser Leu Ile Met Ala Asp TyrCys Leu Asp Phe 1 5 10 15 73 16 PRT homo sapiens 73 Tyr Phe Ala Cys ThrAsn Asn Asp Ser Tyr Leu Phe Cys Leu Asp Phe 1 5 10 15 74 17 PRT homosapiens 74 Asp Asp Met Cys Arg Trp Tyr Pro Phe Ala Ser Phe Tyr Met CysLeu 1 5 10 15 Phe 75 18 PRT homo sapiens 75 Asp Asp His Cys Glu Trp AlaSer Tyr Trp Lys Trp Asp Leu Cys Leu 1 5 10 15 His Asp 76 18 PRT homosapiens 76 Asp Asp Val Cys Glu Asn Ala Asp Phe Ala Trp Leu Gly Trp CysMet 1 5 10 15 His Phe 77 18 PRT homo sapiens 77 Asp Asp Asp Cys Gly TrpIle Gly Phe Ala Asn Phe His Leu Cys Leu 1 5 10 15 His Gly 78 18 PRT homosapiens 78 Phe Asp Asp Cys Gln Thr Ser Trp Phe Gln Gly Phe Trp Leu CysIle 1 5 10 15 Asp Asp 79 18 PRT homo sapiens 79 Phe His Asp Cys Ser TrpGly Pro Trp Gly Pro Trp Glu Ile Cys Thr 1 5 10 15 Arg Leu 80 18 PRT homosapiens 80 Ser Asn Asp Cys Val Trp Leu Gln Phe Trp Gly Gly Asp Met CysPhe 1 5 10 15 Leu Pro 81 18 PRT homo sapiens 81 Asn Ala Asp Cys Glu TrpVal Asn Phe Asn His Val Asp Leu Cys Met 1 5 10 15 Trp Asn 82 18 PRT homosapiens 82 Gly Ser Asp Cys Glu Trp Val Asn Phe Thr Met Phe Gln Met CysIle 1 5 10 15 Ser Asn 83 18 PRT homo sapiens 83 Ala Trp Asp Cys Glu TrpAsn Leu Phe Asp Ser Thr Phe Phe Cys Pro 1 5 10 15 Gly Phe 84 18 PRT homosapiens 84 Leu Tyr Glu Cys Glu Trp Lys Gln Phe Gly Pro Val Glu Met CysLeu 1 5 10 15 Asn Phe 85 18 PRT homo sapiens 85 His Ser Glu Cys Arg TrpGlu Trp Phe Gly Arg Thr Met Ile Cys Met 1 5 10 15 Ser Phe 86 18 PRT homosapiens 86 Ser Gly Glu Cys Asn Trp Gln Gln Phe Ser Gly Trp Glu Ile CysLeu 1 5 10 15 Arg Asp 87 18 PRT homo sapiens 87 Ala Tyr Leu Cys Asp TrpIle Leu Phe Asp Ser Phe Glu Met Cys Leu 1 5 10 15 Ala Pro 88 18 PRT homosapiens 88 Pro Phe Glu Cys Asp Trp Gly Pro Trp Thr Leu Glu Met Leu CysGly 1 5 10 15 Pro Pro 89 15 PRT homo sapiens 89 Arg Gly His Cys Arg AspSer Arg Cys Met Met Asn Ala Pro Gly 1 5 10 15 90 15 PRT homo sapiens 90Arg Ile Gly Cys Arg Asp Ser Arg Cys Asn Trp Trp Ala Pro Gly 1 5 10 15 9112 PRT homo sapiens 91 Arg Gly Phe Cys Arg Asp Ser Ser Cys Ser Phe Pro 15 10 92 12 PRT homo sapiens 92 Arg Gly Trp Cys Leu Asp Ser Arg Cys LysVal Phe 1 5 10 93 16 PRT homo sapiens 93 Phe Leu Phe Cys Arg Leu Ala SerArg Asp Ser Arg Cys Ala Ser Pro 1 5 10 15 94 16 PRT homo sapiens 94 PheAsn Pro Cys Arg Leu Gln Ser Arg Asp Ser Ala Cys Arg Phe Arg 1 5 10 15 9516 PRT homo sapiens 95 Phe Phe Pro Cys Arg Ala Leu Glu Lys Asp Ser ArgCys Ser Phe Phe 1 5 10 15 96 16 PRT homo sapiens 96 His Phe Ser Cys ArgLeu Pro Ser Leu Asp Ser Arg Cys Gln Leu Trp 1 5 10 15 97 12 PRT homosapiens 97 Asn Asp Val Cys Leu Asn Asp Asp Cys Val Tyr Gly 1 5 10 98 12PRT homo sapiens 98 Trp Pro Thr Cys Leu Thr Met Asp Cys Val Tyr Asn 1 510 99 12 PRT homo sapiens 99 His Tyr Asn Cys His Thr Asn Asp Cys Val ValLeu 1 5 10 100 12 PRT homo sapiens 100 His Leu Arg Cys Met Thr Ser AspCys Ile His Phe 1 5 10 101 13 PRT homo sapiens 101 Trp Val Leu Cys PheGlu Trp Glu Asp Cys Asp Glu Lys 1 5 10 102 13 PRT homo sapiens 102 TyrGlu Tyr Cys Phe Glu Trp Glu Gln Cys Trp Glu Lys 1 5 10 103 13 PRT homosapiens 103 Gly Ile Phe Cys Phe Glu Trp Glu Thr Cys Tyr Gln Ala 1 5 10104 11 PRT homo sapiens 104 Pro Gln Phe Cys Phe Glu Trp Glu Pro Cys Phe1 5 10 105 13 PRT homo sapiens 105 Ile Gly Phe Cys Phe Glu Trp Glu ValCys Tyr Glu Gly 1 5 10 106 13 PRT homo sapiens 106 Ser Ile Tyr Cys PheAsp Trp Glu Asp Cys Trp Asp Glu 1 5 10 107 13 PRT homo sapiens 107 TyrAsp Trp Cys Phe Asp Trp Glu Gln Cys Trp Asp Gln 1 5 10 108 13 PRT homosapiens 108 Val Gly Phe Cys Phe Asp Trp Glu Pro Cys Asp Glu Leu 1 5 10109 13 PRT homo sapiens 109 Met Asp Phe Cys Phe Asp Trp Glu Glu Cys TrpThr Asn 1 5 10 110 13 PRT homo sapiens 110 Asn Ile Phe Cys Phe Asp TrpGlu Pro Cys His Phe Gly 1 5 10 111 13 PRT homo sapiens 111 Phe Glu IleCys Phe Asp Trp Glu Val Cys His Glu Gln 1 5 10 112 13 PRT homo sapiens112 Asp Tyr Leu Cys Phe Asp Trp Glu Ala Cys Trp Leu Ser 1 5 10 113 13PRT homo sapiens 113 Tyr Ala Met Cys Phe Asp Trp Asp Glu Cys Phe Leu Gly1 5 10 114 14 PRT homo sapiens MISC_FEATURE (2)..(2) X equals any aminoacid 114 Trp Xaa Trp Cys Phe Glu Trp Glu Asp Trp Cys Leu Val Glu 1 5 10115 14 PRT homo sapiens 115 Tyr Gln Phe Cys Phe Asp Trp Glu Thr Thr CysTrp Leu Asp 1 5 10 116 14 PRT homo sapiens 116 Val Tyr Phe Cys Phe AspTrp Glu Gln Asp Cys Asp Glu Met 1 5 10 117 14 PRT homo sapiens 117 PheGln Leu Cys Phe Asp Trp Glu Glu Glu Cys Glu Glu Ser 1 5 10 118 13 PRThomo sapiens 118 Trp Ala Val Cys Phe Asp Trp Glu Asn Cys Gly Asp Lys 1 510 119 14 PRT homo sapiens 119 Trp Gln Phe Cys Phe Asp Trp Asp Leu AsnCys Asp Leu Arg 1 5 10 120 18 PRT homo sapiens 120 Tyr Trp Phe Cys PheAsp Trp Glu Glu Asp Ala Asn Gly His Cys Gly 1 5 10 15 Gly Asn 121 18 PRThomo sapiens 121 Phe Leu Leu Cys Phe Asp Trp Asp Ile Asp Trp Glu Tyr GlyCys Gln 1 5 10 15 His His 122 14 PRT homo sapiens 122 Tyr Glu Glu CysHis Trp Arg Pro Met Ala Cys Ser Thr His 1 5 10 123 14 PRT homo sapiens123 Trp Glu Val Cys His Trp Ala Pro Met Met Cys Lys His Gly 1 5 10 12414 PRT homo sapiens 124 Tyr Glu Phe Cys His Tyr Ala Pro Gln Glu Cys LysHis Met 1 5 10 125 14 PRT homo sapiens MISC_FEATURE (1)..(1) X equalsany amino acid 125 Xaa Lys Glu Cys Lys Phe Gly Tyr Ser Xaa Cys Leu AlaTrp 1 5 10 126 14 PRT homo sapiens 126 Gln Lys Glu Cys Lys Phe Gly TyrPro His Cys Leu Pro Trp 1 5 10 127 13 PRT homo sapiens 127 Glu His AsnCys Thr Trp Trp Asn Pro Cys Trp Thr Thr 1 5 10 128 13 PRT homo sapiens128 Met Asp His Cys Thr Trp Tyr Gln Pro Cys Val Leu Lys 1 5 10 129 13PRT homo sapiens 129 Trp Asp His Cys Asn Trp Ala His Pro Cys Ser Arg Lys1 5 10 130 14 PRT homo sapiens 130 Ser Asp Trp Cys Gly Thr Trp Asn AsnPro Cys Phe His Gln 1 5 10 131 18 PRT homo sapiens 131 Arg Tyr Leu CysLeu Pro Gln Arg Asp Lys Pro Trp Lys Phe Cys Asn 1 5 10 15 Trp Phe 132 18PRT homo sapiens 132 Arg Leu His Cys Lys Pro Gln Arg Gln Ser Pro Trp MetLys Cys Gln 1 5 10 15 His Leu 133 18 PRT homo sapiens 133 Tyr Ser HisCys Ser Pro Leu Arg Tyr Tyr Pro Trp Trp Lys Cys Thr 1 5 10 15 Tyr Pro134 18 PRT homo sapiens 134 Leu His Ala Cys Arg Pro Val Arg Gly Asp ProTrp Trp Ala Cys Thr 1 5 10 15 Leu Gly 135 18 PRT homo sapiens 135 GlyPhe Thr Cys Ser Pro Ile Arg Met Phe Pro Trp Phe Arg Cys Asp 1 5 10 15Leu Gly 136 18 PRT homo sapiens 136 Phe Ser Pro Cys Lys Ala Leu Arg HisSer Pro Trp Trp Val Cys Pro 1 5 10 15 Ser Gly 137 2920 DNA homo sapiensmisc_feature (1707)..(1707) n equals any amino acid 137 gtggatgtgatcttggctcc ccggggacga tgtcagctct tcctggctcc ttctcagcct 60 tgttgctgtaactgctgctc agtccaccat tgaggaacag gccaagacat ttttgggaca 120 agtttaaccacgaagccgaa gacctgttct atcaaagttc acttgcttct tggaattata 180 acaccaatattactgaagag aatgtccaaa acatgaataa tgctggggac aaatggtctg 240 cctttttaaaggaacagtcc acacttgccc aaatgtatcc actacaagaa attcagaatc 300 tcacagtcaagcttcagctg caggctcttc agcaaaatgg gtcttcagtg ctctcagaag 360 acaagagcaaacggttgaac acaattctaa atacaatgag caccatctac agtactggaa 420 aagtttgtaacccagataat ccacaagaat gcttattact tgaaccaggt ttgaatgaaa 480 taatggcaaacagtttagac tacaatgaga ggctctgggc ttgggaaagc tggagatctg 540 aggtcggcaagcagctgagg ccattatatg aagagtatgt ggtcttgaaa aatgagatgg 600 caagagcaaatcattatgag gactatgggg attattggag aggagactat gaagtaaatg 660 gggtagatggctatgactac agccgcggcc agttgattga agatgtggaa catacctttg 720 aagagattaaaccattatat gaacatcttc atgcctatgt gaggccaaag ttgatgaatg 780 cctatccttcctatatcagt ccaattggat gcctccctgc tcatttgctt ggtgatatgt 840 ggggtagattttggacaaat ytgtacwstt tgacagttcc ctttggacag aaaccaaaca 900 tagatgttactgatgcaatg gtggaccagr cctgggatgc acagagaata ttcaaggagg 960 ccgagaagttctttgtatct gttggtcttc ctaatatgac tcaaggattc tgggaaaatt 1020 ccatgctaacggacccagga aatgttcaga aagcagtctg ccatcccaca gcttgggacc 1080 tggggaagggcgacttcagg atccttatgt gcacaaaggt gacaatggac gacttcctga 1140 cagctcatcatgagatgggg catatccagt atgatatggc atatgctgca caaccttttc 1200 tgctaagaaatggagctaat gaaggattcc atgaagctgt tggggaaatc atgtcacttt 1260 ctgcagccacacctaagcat ttaaaatcca ttggtcttct gtcacccgat tttcaagaag 1320 acaatgaaacagaaataaac ttcctgctca aacaagcact cacgattgtt gggactctgc 1380 catttacttacatgttagag aagtggaggt ggatggtctt taaaggggaa attcccaaag 1440 accagtggatgaaaaagtgg tgggagatga agcgagagat agttggggtg gtggaacctg 1500 tgccccatgatgaaacatac tgtgaccccg catctctgtt ccatgtttct aatgattact 1560 cattcattcgatattacaca aggacccttt accaattcca gtttcaagaa gcactttgtc 1620 aagcagctaaacatgaaggc cctctgcaca aatgtgacat ctcaaactct acagaagctg 1680 gacagaaactgttcaatatg ctgaggnttg gaaaatcaga accctggacc ctagcattgg 1740 aaaatgttgtaggagcaaag aacatgaatg taaggccact gctcaactac tttgagccct 1800 tatttacctggctgaaagac cagaacaaga attcttttgt gggatggagt accgactgga 1860 gtccatatgcagaccaaagc atcaaagtga ggataagcct aaaatcagct cttggagata 1920 aagcatatgaatggaacgac aatgaaatgt acctgttccg atcatctgtt gcatatgcta 1980 tgaggcagtactttttaaaa gtaaaaaatc agatgattct ttttggggag gaggatgtgc 2040 gagtggctaatttgaaacca agaatctcct ttaatttctt tgtcactgca cctaaaaatg 2100 tgtctgatatcattcctaga actgaagttg aaaaggccat caggatgtcc cggagccgta 2160 tcaatgatgctttccgtctg aatgacgaca gcctagagtt tctggggata cagccaacac 2220 ttggacctcctaaccagccc cctgtttcca tatggctgat tgtttttgga gttgtgatgg 2280 gagtgatagtggttggcatt gtcatcctga tcttcactgg gatcagagat cggaagaagg 2340 gcctgtaaatggaattcctg cattgctcta accatgtaca accttggact tagcttttac 2400 ctgtaactggcttctgagag acaaagagga gaaaccttca ctcctagtac acctattaca 2460 gctgcagaggtagaggagac agttgcagaa ctagttacaa tgacgataag aaacaatact 2520 ttgttattccatagcacctt taatgttcat gtgtattatc tcagctagcc ttgaaccgcc 2580 taagtaaggtgatgaggacg ggtttaagcc ccactgatat tttaaaagcc cagagaaaag 2640 tgttcgttcctctactaacc tgttctttta gagcagggat ctgcatacta ggcctgcagc 2700 cnaaatgagtaggtagccca ctacctattt ttgtatagcc agagggctna gaatggnttt 2760 tacattttaagtggttttac atttaagnnc aaaagaagga taatatttca tgacaagtna 2820 aaattatatgaactnaaaat tgtatgaatt ttatgnattt atatttcagt attcataatt 2880 aaagttttattgaactacaa aaaaaaaaaa aaaaaaaaaa 2920 138 711 PRT homo sapiensMISC_FEATURE (219)..(219) Xaa equals any amino acid 138 Met Asn Asn AlaGly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser 1 5 10 15 Thr Leu AlaGln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val 20 25 30 Lys Leu GlnLeu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser 35 40 45 Glu Asp LysSer Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr 50 55 60 Ile Tyr SerThr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys 65 70 75 80 Leu LeuLeu Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp 85 90 95 Tyr AsnGlu Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly 100 105 110 LysGln Leu Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu 115 120 125Met Ala Arg Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly 130 135140 Asp Tyr Glu Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln 145150 155 160 Leu Ile Glu Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro LeuTyr 165 170 175 Glu His Leu His Ala Tyr Val Arg Pro Lys Leu Met Asn AlaTyr Pro 180 185 190 Ser Tyr Ile Ser Pro Ile Gly Cys Leu Pro Ala His LeuLeu Gly Asp 195 200 205 Met Trp Gly Arg Phe Trp Thr Asn Leu Tyr Xaa LeuThr Val Pro Phe 210 215 220 Gly Gln Lys Pro Asn Ile Asp Val Thr Asp AlaMet Val Asp Gln Xaa 225 230 235 240 Trp Asp Ala Gln Arg Ile Phe Lys GluAla Glu Lys Phe Phe Val Ser 245 250 255 Val Gly Leu Pro Asn Met Thr GlnGly Phe Trp Glu Asn Ser Met Leu 260 265 270 Thr Asp Pro Gly Asn Val GlnLys Ala Val Cys His Pro Thr Ala Trp 275 280 285 Asp Leu Gly Lys Gly AspPhe Arg Ile Leu Met Cys Thr Lys Val Thr 290 295 300 Met Asp Asp Phe LeuThr Ala His His Glu Met Gly His Ile Gln Tyr 305 310 315 320 Asp Met AlaTyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn 325 330 335 Glu GlyPhe His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala 340 345 350 ThrPro Lys His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln 355 360 365Glu Asp Asn Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr 370 375380 Ile Val Gly Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp 385390 395 400 Met Val Phe Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys LysTrp 405 410 415 Trp Glu Met Lys Arg Glu Ile Val Gly Val Val Glu Pro ValPro His 420 425 430 Asp Glu Thr Tyr Cys Asp Pro Ala Ser Leu Phe His ValSer Asn Asp 435 440 445 Tyr Ser Phe Ile Arg Tyr Tyr Thr Arg Thr Leu TyrGln Phe Gln Phe 450 455 460 Gln Glu Ala Leu Cys Gln Ala Ala Lys His GluGly Pro Leu His Lys 465 470 475 480 Cys Asp Ile Ser Asn Ser Thr Glu AlaGly Gln Lys Leu Phe Asn Met 485 490 495 Leu Arg Xaa Gly Lys Ser Glu ProTrp Thr Leu Ala Leu Glu Asn Val 500 505 510 Val Gly Ala Lys Asn Met AsnVal Arg Pro Leu Leu Asn Tyr Phe Glu 515 520 525 Pro Leu Phe Thr Trp LeuLys Asp Gln Asn Lys Asn Ser Phe Val Gly 530 535 540 Trp Ser Thr Asp TrpSer Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg 545 550 555 560 Ile Ser LeuLys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp 565 570 575 Asn GluMet Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln 580 585 590 TyrPhe Leu Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp 595 600 605Val Arg Val Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val 610 615620 Thr Ala Pro Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu 625630 635 640 Lys Ala Ile Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe ArgLeu 645 650 655 Asn Asp Asp Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr LeuGly Pro 660 665 670 Pro Asn Gln Pro Pro Val Ser Ile Trp Leu Ile Val PheGly Val Val 675 680 685 Met Gly Val Ile Val Val Gly Ile Val Ile Leu IlePhe Thr Gly Ile 690 695 700 Arg Asp Arg Lys Lys Gly Leu 705 710 139 17PRT homo sapiens 139 Leu Ile Val Phe Gly Val Val Met Gly Val Ile Val ValGly Ile Val 1 5 10 15 Ile 140 681 PRT homo sapiens MISC_FEATURE(219)..(219) Xaa equals any amino acid 140 Met Asn Asn Ala Gly Asp LysTrp Ser Ala Phe Leu Lys Glu Gln Ser 1 5 10 15 Thr Leu Ala Gln Met TyrPro Leu Gln Glu Ile Gln Asn Leu Thr Val 20 25 30 Lys Leu Gln Leu Gln AlaLeu Gln Gln Asn Gly Ser Ser Val Leu Ser 35 40 45 Glu Asp Lys Ser Lys ArgLeu Asn Thr Ile Leu Asn Thr Met Ser Thr 50 55 60 Ile Tyr Ser Thr Gly LysVal Cys Asn Pro Asp Asn Pro Gln Glu Cys 65 70 75 80 Leu Leu Leu Glu ProGly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp 85 90 95 Tyr Asn Glu Arg LeuTrp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly 100 105 110 Lys Gln Leu ArgPro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu 115 120 125 Met Ala ArgAla Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly 130 135 140 Asp TyrGlu Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln 145 150 155 160Leu Ile Glu Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr 165 170175 Glu His Leu His Ala Tyr Val Arg Pro Lys Leu Met Asn Ala Tyr Pro 180185 190 Ser Tyr Ile Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp195 200 205 Met Trp Gly Arg Phe Trp Thr Asn Leu Tyr Xaa Leu Thr Val ProPhe 210 215 220 Gly Gln Lys Pro Asn Ile Asp Val Thr Asp Ala Met Val AspGln Xaa 225 230 235 240 Trp Asp Ala Gln Arg Ile Phe Lys Glu Ala Glu LysPhe Phe Val Ser 245 250 255 Val Gly Leu Pro Asn Met Thr Gln Gly Phe TrpGlu Asn Ser Met Leu 260 265 270 Thr Asp Pro Gly Asn Val Gln Lys Ala ValCys His Pro Thr Ala Trp 275 280 285 Asp Leu Gly Lys Gly Asp Phe Arg IleLeu Met Cys Thr Lys Val Thr 290 295 300 Met Asp Asp Phe Leu Thr Ala HisHis Glu Met Gly His Ile Gln Tyr 305 310 315 320 Asp Met Ala Tyr Ala AlaGln Pro Phe Leu Leu Arg Asn Gly Ala Asn 325 330 335 Glu Gly Phe His GluAla Val Gly Glu Ile Met Ser Leu Ser Ala Ala 340 345 350 Thr Pro Lys HisLeu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln 355 360 365 Glu Asp AsnGlu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr 370 375 380 Ile ValGly Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp 385 390 395 400Met Val Phe Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp 405 410415 Trp Glu Met Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His 420425 430 Asp Glu Thr Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp435 440 445 Tyr Ser Phe Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe GlnPhe 450 455 460 Gln Glu Ala Leu Cys Gln Ala Ala Lys His Glu Gly Pro LeuHis Lys 465 470 475 480 Cys Asp Ile Ser Asn Ser Thr Glu Ala Gly Gln LysLeu Phe Asn Met 485 490 495 Leu Arg Xaa Gly Lys Ser Glu Pro Trp Thr LeuAla Leu Glu Asn Val 500 505 510 Val Gly Ala Lys Asn Met Asn Val Arg ProLeu Leu Asn Tyr Phe Glu 515 520 525 Pro Leu Phe Thr Trp Leu Lys Asp GlnAsn Lys Asn Ser Phe Val Gly 530 535 540 Trp Ser Thr Asp Trp Ser Pro TyrAla Asp Gln Ser Ile Lys Val Arg 545 550 555 560 Ile Ser Leu Lys Ser AlaLeu Gly Asp Lys Ala Tyr Glu Trp Asn Asp 565 570 575 Asn Glu Met Tyr LeuPhe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln 580 585 590 Tyr Phe Leu LysVal Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp 595 600 605 Val Arg ValAla Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val 610 615 620 Thr AlaPro Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu 625 630 635 640Lys Ala Ile Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu 645 650655 Asn Asp Asp Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro 660665 670 Pro Asn Gln Pro Pro Val Ser Ile Trp 675 680 141 3396 DNA homosapiens 141 gaattcggct tccatcctaa tacgactcac tatagggctc gagcggccgcccggggcagg 60 tatcttggct cacaggggac gatgtcaagc tcttcctggc tccttctcagccttgttgct 120 gtaactgctg ctcagtccac cattgaggaa caggccaaga catttttggacaagtttaac 180 cacgaagccg aagacctgtt ctatcaaagt tcacttgctt cttggaattataacaccaat 240 attactgaag agaatgtcca aaacatgaat aatgctgggg acaaatggtctgccttttta 300 aaggaacagt ccacacttgc ccaaatgtat ccactacaag aaattcagaatctcacagtc 360 aagcttcagc tgcaggctct tcagcaaaat gggtcttcag tgctctcagaagacaagagc 420 aaacggttga acacaattct aaatacaatg agcaccatct acagtactggaaaagtttgt 480 aacccagata atccacaaga atgcttatta cttgaaccag gtttgaatgaaataatggca 540 aacagtttag actacaatga gaggctctgg gcttgggaaa gctggagatctgaggtcggc 600 aagcagctga ggccattata tgaagagtat gtggtcttga aaaatgagatggcaagagca 660 aatcattatg aggactatgg ggattattgg agaggagact atgaagtaaatggggtagat 720 ggctatgact acagccgcgg ccagttgatt gaagatgtgg aacatacctttgaagagatt 780 aaaccattat atgaacatct tcatgcctat gtgagggcaa agttgatgaatgcctatcct 840 tcctatatca gtccaattgg atgcctccct gctcatttgc ttggtgatatgtggggtaga 900 ttttggacaa atctgtactc tttgacagtt ccctttggac agaaaccaaacatagatgtt 960 actgatgcaa tggtggacca ggcctgggat gcacagagaa tattcaaggaggccgagaag 1020 ttctttgtat ctgttggtct tcctaatatg actcaaggat tctgggaaaattccatgcta 1080 acggacccag gaaatgttca gaaagcagtc tgccatccca cagcttgggacctggggaag 1140 ggcgacttca ggatccttat gtgcacaaag gtgacaatgg acgacttcctgacagctcat 1200 catgagatgg ggcatatcca gtatgatatg gcatatgctg cacaaccttttctgctaaga 1260 aatggagcta atgaaggatt ccatgaagct gttggggaaa tcatgtcactttctgcagcc 1320 acacctaagc atttaaaatc cattggtctt ctgtcacccg attttcaagaagacaatgaa 1380 acagaaataa acttcctgct caaacaagca ctcacgattg ttgggactctgccatttact 1440 tacatgttag agaagtggag gtggatggtc tttaaagggg aaattcccaaagaccagtgg 1500 atgaaaaagt ggtgggagat gaagcgagag atagttgggg tggtggaacctgtgccccat 1560 gatgaaacat actgtgaccc cgcatctctg ttccatgttt ctaatgattactcattcatt 1620 cgatattaca caaggaccct ttaccaattc cagtttcaag aagcactttgtcaagcagct 1680 aaacatgaag gccctctgca caaatgtgac atctcaaact ctacagaagctggacagaaa 1740 ctgttcaata tgctgaggct tggaaaatca gaaccctgga ccctagcattggaaaatgtt 1800 gtaggagcaa agaacatgaa tgtaaggcca ctgctcaact actttgagcccttatttacc 1860 tggctgaaag accagaacaa gaattctttt gtgggatgga gtaccgactggagtccatat 1920 gcagaccaaa gcatcaaagt gaggataagc ctaaaatcag ctcttggagataaagcatat 1980 gaatggaacg acaatgaaat gtacctgttc cgatcatctg ttgcatatgctatgaggcag 2040 tactttttaa aagtaaaaaa tcagatgatt ctttttgggg aggaggatgtgcgagtggct 2100 aatttgaaac caagaatctc ctttaatttc tttgtcactg cacctaaaaatgtgtctgat 2160 atcattccta gaactgaagt tgaaaaggcc atcaggatgt cccggagccgtatcaatgat 2220 gctttccgtc tgaatgacaa cagcctagag tttctgggga tacagccaacacttggacct 2280 cctaaccagc cccctgtttc catatggctg attgtttttg gagttgtgatgggagtgata 2340 gtggttggca ttgtcatcct gatcttcact gggatcagag atcggaagaagaaaaataaa 2400 gcaagaagtg gagaaaatcc ttatgcctcc atcgatatta gcaaaggagaaaataatcca 2460 ggattccaaa acactgatga tgttcagacc tccttttaga aaaatctatgtttttcctct 2520 tgaggtgatt ttgttgtatg taaatgttaa tttcatggta tagaaaatataagatgataa 2580 agatatcatt aaatgtcaaa actatgactc tgttcagaaa aaaaattgtccaaagacaac 2640 atggccaagg agagagcatc ttcattgaca ttgctttcag tatttatttctgtctctgga 2700 tttgacttct gttctgtttc ttaataagga ttttgtatta gagtatattagggaaagtgt 2760 gtatttggtc tcacaggctg ttcagggata atctaaatgt aaatgtctgttgaatttctg 2820 aagttgaaaa caaggatata tcattggagc aagtgttgga tcttgtatggaatatggatg 2880 gatcacttgt aaggacagtg cctgggaact ggtgtagctg caaggattgagaatggcatg 2940 cattagctca ctttcattta atccattgtc aaggatgaca tgctttcttcacagtaactc 3000 agttcaagta ctatggtgat ttgcctacag tgatgtttgg aatcgatcatgctttcttca 3060 aggtgacagg tctaaagaga gaagaatcca gggaacaggt agaggacattgctttttcac 3120 ttccaaggtg cttgatcaac atctccctga caacacaaaa ctagagccaggggcctccgt 3180 gaactcccag agcatgcctg atagaaactc atttctactg ttctctaactgtggagtgaa 3240 tggaaattcc aactgtatgt tcaccctctg aagtgggtac ccagtctcttaaatcttttg 3300 tatttgctca cagtgtttga gcagtgctga gcacaaagca gacactcaataaatgctaga 3360 tttacacact caaaaaaaaa aaaaaagggc ggccgc 3396 142 805 PRThomo sapiens 142 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala ValThr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe LeuAsp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser LeuAla Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln AsnMet Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln SerThr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu ThrVal Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val LeuSer Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr MetSer Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn ProGln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met AlaAsn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu SerTrp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu GluTyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr GluAsp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly ValAsp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val GluHis Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 HisAla Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val GlyLeu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met LeuThr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr AlaTrp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr LysVal Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly HisIle Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu ArgAsn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu IleMet Ser Leu Ser Ala Ala Thr Pro Lys 405 410 415 His Leu Lys Ser Ile GlyLeu Leu Ser Pro Asp Phe Gln Glu Asp Asn 420 425 430 Glu Thr Glu Ile AsnPhe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly 435 440 445 Thr Leu Pro PheThr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe 450 455 460 Lys Gly GluIle Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met 465 470 475 480 LysArg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr 485 490 495Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe 500 505510 Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala 515520 525 Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile530 535 540 Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu ArgLeu 545 550 555 560 Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn ValVal Gly Ala 565 570 575 Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr PheGlu Pro Leu Phe 580 585 590 Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser PheVal Gly Trp Ser Thr 595 600 605 Asp Trp Ser Pro Tyr Ala Asp Gln Ser IleLys Val Arg Ile Ser Leu 610 615 620 Lys Ser Ala Leu Gly Asp Lys Ala TyrGlu Trp Asn Asp Asn Glu Met 625 630 635 640 Tyr Leu Phe Arg Ser Ser ValAla Tyr Ala Met Arg Gln Tyr Phe Leu 645 650 655 Lys Val Lys Asn Gln MetIle Leu Phe Gly Glu Glu Asp Val Arg Val 660 665 670 Ala Asn Leu Lys ProArg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro 675 680 685 Lys Asn Val SerAsp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile 690 695 700 Arg Met SerArg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn 705 710 715 720 SerLeu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln 725 730 735Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val 740 745750 Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg 755760 765 Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile770 775 780 Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr AspAsp 785 790 795 800 Val Gln Thr Ser Phe 805 143 10 PRT homo sapiens 143Asp Arg Val Tyr Ile His Pro Phe His Leu 1 5 10 144 8 PRT homo sapiens144 Asp Arg Val Tyr Ile His Pro Phe 1 5 145 9 PRT homo sapiens 145 AspArg Val Tyr Ile His Pro Phe His 1 5 146 8 PRT homo sapiens 146 Pro GlyPro Glu Gly Gly Gly Lys 1 5 147 8 PRT homo sapiens 147 Asp Tyr Lys AspAsp Asp Asp Lys 1 5 148 17 PRT homo sapiens 148 Met Leu Gln Asn Ser AlaVal Leu Leu Leu Leu Val Ile Ser Ala Ser 1 5 10 15 Ala 149 22 PRT homosapiens 149 Met Pro Thr Trp Ala Trp Trp Leu Phe Leu Val Leu Leu Leu AlaLeu 1 5 10 15 Trp Ala Pro Ala Arg Gly 20 150 733 DNA homo sapiens 150gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120tctcccggac tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660acaaccacta cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720gactctagag gat 733 151 34 DNA homo sapiens 151 atggatgatc agccatcatgtcaagctctt cctg 34 152 35 DNA homo sapiens 152 gtatgctcta gattaggaaacagggggctg gttag 35 153 12 PRT homo sapiens MISC_FEATURE (1)..(4) Xequals any amino acid 153 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa XaaXaa 1 5 10 154 13 PRT homo sapiens MISC_FEATURE (1)..(4) X equals anyamino acid 154 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 510 155 14 PRT homo sapiens MISC_FEATURE (1)..(4) X equals any amino acid155 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15615 PRT homo sapiens MISC_FEATURE (1)..(3) X equals any amino acid 156Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15157 16 PRT homo sapiens MISC_FEATURE (1)..(3) X equals any amino acid157 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 510 15 158 18 PRT homo sapiens MISC_FEATURE (1)..(3) X equals any aminoacid 158 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa1 5 10 15 Xaa Xaa

What is claimed is:
 1. A method of increasing vasoconstrictioncomprising administering to an individual a therapeutically effectiveamount of angiotensin II in combination with angiotensin 1-9.
 2. Amethod of ameliorating a disorder resulting from low blood pressurecomprising administering a therapeutically effective amount ofangiotensin II in combination with angiotensin 1-9.
 3. The method ofclaim 2 wherein the disorder is hypotension.
 4. The method of claim 2wherein the disorder is shock.
 5. The method of claim 2 wherein thedisorder is syncope.